Canine Brucella

DiagXotics® , Inc.

DiagXotics Test Kits


 
Test Kits for SHRIMP
Format Disease
Dot Blot BP; HPV;, IHHNV; MBV; NHP; WSSV
In Situ BP; HPV; IHHNV; NHP; TSV; WSSV; YHV
ImmunoDot TSV; WSSV
ImmunoHistochemistry TSV; WSSV
ImmunoSquash WSSV
Spot-On Monitor the health of your shrimp in just 15 minutes
Simplex Primer Kits for PCR BP; HPV; MBV; NHP; IHHNV; WSSV
Duplex Primer Kits for PCR IHHNV with WSSV
Multiplex Primer Kits for PCR
WSSV with IHHNV,NHP, and HPV;
WSSV with IHHNV and BP;
WSSV with IHHNV,and HPV

Primer Kits for RT-PCR TSV; YHV
Electrophoresis Kit for PCR
DNA Extraction Kits
Test Kits for FINFISH
Format Disease
Elisa Field BKD; Furunculosis; IPNV
Elisa Laboratory BKD; Furunculosis; IPNV
Simplex PCR Test Kits BKD; SRS
ICA BKD; IPNV
Test Kits for Ruminants
Format Disease
Elisa Laboratory Bluetongue Virus
 
Test Kits for MOUSE
Format Disease
Primer Kits for RT-PCR Mouse Hepatitis Virus


"Providing Reliable Results to Protect Dogs"

DIAGNOSTIC TEST KITS FOR ANIMAL HEALTH

 
DiagXotics is a leader in the field of animal health, manufactures comprehensive disease diagnostic kits and vaccine-like products for the detection and management of animal diseases. Using our state of the art technology, and drawing upon our 12 years of experience in disease diagnostics, DiagXotics provides the tools to protect our clients' investment from financial devastation.
 
Available Products:

Canine Brucella Test Kit

Shrimp Diagnostic Test Kits - Primer Kits for PCR
Shrimp Diagnostic Test Kits - Primer Kits for RT-PCR
 
 
At this time, only the ShrimpCare PCR and RT-PCR Primer Diagnostic Kits are in stock and available. However, DiagXotics, Inc. has developed several other quality products that may be available again soon. We invite to inquire about these products by joining our mailing list. You will be notified as soon as these products become available again.




 
 

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Intuitive

We create easy-to-use products.

Adaptable

We build modular solutions.

Durable

We craft long-lasting goods.

DiagXotics® , Inc.
"Providing Reliable Results to Protect Your Investment"

PRICING FOR DIAGNOSTIC KITS

 
All prices are quoted in U.S. Dollars and subject to change without notice.
 
SHRIMP
Test Format
Tests Per Kit
Basic Kit
Regular Kit
Order
Simplex Primer Kit for PCR 100* $225.00 $650.00
Duplex Primer Kit for PCR 100* $260.00 $750.00
Multiplex Primer Kit for PCR 100* $350.00 $1,500.00
Primer Kit for RT-PCR 100* $225.00 $1,500.00

FAQ (Frequently Asked Questions)

Be sure to read all instructions

It is very important to read each instruction booklet thoroughly before beginning each test. Make note of the temperature requirements of each reagent and of each incubation step, and be sure that each temperature is reached before beginning. Also check for any additional equipment, materials, and reagents that are needed but not supplied with the kit before beginning the test. If there are any questions concerning the protocol, please contact your distributor or DiagXotics for further assistance.

 
What is the shelf life for each kit?

The kits are guaranteed to have a shelf life of 1 year from date of purchase provided each reagent within the kit is stored at its optimal temperature. Each kit insert booklet will list the items included with the correct storage temperature for each.


What are the initial colors of various substrates provided with the kits?

ABTS: This solution has a light greenish tint. It is ok for use. When the solution positively reacts or becomes contaminated, it will turn dark green. Do not use the solution if it is dark green at the start of the test. Hydrogen peroxide must be added to the ABTS in order for a reaction to take place.

Development Solution: This solution should be yellow to clear at the beginning of each test. Over time the solution may slightly precipitate along the inside of the bottle. When checking to see if the solution is still good, a small amount should be removed from the bottle and viewed. If it is yellow, then it is ok for use. Because of the possible precipitation, looking into the bottle may cause an inaccurate assumption that the solution itself has turned purple. This is why a small sample should be removed and viewed.

K-Blue Max: This solution should be clear or it may have a slight bluish tint. When this solution reacts positively or if it becomes contaminated, it will turn a dark blue. If it is dark blue at the beginning of the test, it should not be used.

 
What if my kits are delayed in shipment? Will they still work?

Shelf life studies have been conducted in-house to ensure that the length of time it takes for an item to be shipped and clear customs has no effect on its performance. However, upon receipt of the kit, each item must be placed at its optimal temperature to prevent degradation. DiagXotics cannot guarantee any item that has not been stored at the correct temperature.

Items that are stored frozen are shipped with an additional ice pack to keep them as cold as possible for as long as possible. If the ice pack is thawed upon arrival this will not harm the reagents. These reagents must be refrozen upon receipt of the kit.

 
How do I know if the reagents are still working properly?

The kits come with internal controls. These controls must be used during each run to insure that the test has worked properly. If there is a question about any of the reagents, the controls can be run to test the activity. If the controls work then the reagents can continue to be used to test samples. However if the controls do not work or are weaker than they should be, the kit should not be used to test samples.


How should I store the reagents?

Correct storage temperatures for each item sent will be printed either on the label of each item or listed in the product insert. Each item must be stored at its optimal temperature in order for it to work and continue to work for the duration of its shelf life. Reagents should never be stored in direct sunlight.

 
Which kits are DNA based and which kits are Monoclonal Antibody based?

Kits available for the detection of DNA/RNA:

In Situ
Dot Blot
PCR Primer Kits

Kits that contain Monoclonal antibodies:

ImmunoDot
Immunohistochemistry
ELISA
ICA
ImmunoSquash


What is the difference between a polyclonal antibody and a monoclonal antibody?

Polyclonal Antibodies contain a mixture of antibodies that recognize various regions of an antigen. Because of these variations it is not possible to exactly replicate a batch of polyclonal antibody. Each subsequent batch produced will vary from the previous batch.

Monoclonal Antibodies are those produced by taking a single antibody-producing cell and fusing it with a myloma cell to give it unlimited replication. This allows for multiple batches to be produced that are exactly the same as previously produced batches. Using the same antibody allows for reproducibility of the test and for increased specificity in the detection of an antigen.


How do I report a problem?

If there is a problem found with one of the kits, please notify your distributor or DiagXotics immediately. Before reporting a problem, please obtain as much information about the kit as possible including lot numbers located on the reagent labels, serial numbers, and manufacture date of the kit that is located on the outer carton.

 
What is Spot-On?

Spot-On is a rapid non-specific test that is used to assess the overall health of shrimp. It is similar to the use of a thermometer in humans in that it will determine if something is wrong with the animal but it will not identify the cause of the problem.


What are Ready To Go Beads?

The Ready To Go Beads contain lyophilized reagents that are necessary for the replication of DNA in PCR. This eliminates the need to prepare a liquid master mix. It also serves to increase conformity between tubes run in the same batch and multiple batches.


What is the sample source for the ImmunoSquash Test Kit?

The ImmunoSquash can only be run using gill tissue. The test requires at least 1 gill filament from the animal with a maximum of 5 gill filaments per tubes.


What is the sample source for Dot Blot?

The sample source for the Dot Blot test kit varies depending on the pathogen that is being tested for. Testing for pathogens that are systemic can be done lethally or non-lethally. Systemic pathogens can be detected in hemolymph, pleopods, gill tissue, epidermis, etc.

When testing for pathogens that are non-systemic the target tissue for the pathogen must be used as the sample source. If any other tissues, or hemolymph are used it reduces the chance of detecting the antigen. In a case such as this, if the result is negative it cannot be assumed that the animal is not harboring the pathogen in the target tissue.


What is the sample source for ImmunoDot?

The ImmunoDot for both TSV and WSSV use hemolymph as the sample source. Tissue samples would clog the membrane interfering with the testing procedure.


Are positively charged slides necessary?

When running an In Situ the tissue sections must be fixed to positively charged slides. If regular slides are used the tissue will wash off the slides during testing.

The Immunohistochemistry test is not as harsh as the In Situ so tissue sections can be fixed onto non-positively charged slides. However, positively charged slides should be used when possible to prevent loss of tissue.


Can I pool samples?

It is best not to pool samples during testing. One test should represent one animal. This will decrease the chance of false negative results caused by diluting a positive sample with numerous other samples that may be negative.

If pooling needs to be done for economic reasons, no more than 5 samples of equal volume or size should be pooled..

Our References

Tips

Troubleshooting


Format
ELISA
Dot Blot
In Situ/Immunohistochemistry
 
ELISA Test Format for lab or field - Tips
(Enzyme Linked Immunosorbant Assay)
1. All reagents must be at room temperature before use

Remove the solutions from the refrigerator ahead of time and place them in an area away from direct sunlight, a/c vents and avoid placing them on top of cold counters cold. Each solution should be warm to the touch before use. Using reagents that are cold will cause a substantial decrease in the end reading.

2. Adding solution to wells/tubes

It is important to add the same amount of solution/sample to each well or tube. When pipetting solutions into wells, check the pipette tip for bubbles. Any bubbles within the pipette will cause an inaccurate volume to be dispensed. Variations between volumes added to wells will caused variations in the signal when the plate is read. When dispensing solutions from a dropper bottle, the bottle should be held completely upright and gently squeezed. This will help to prevent bubbles, and ensure that the same volume is dispensed each time.

3. Avoiding cross contamination

Contamination can occur between samples, and between reagents. To prevent this, clean pipette tips should be used for each new sample and/or reagent. If the tip of the pipette comes in contact with a well, it should be discarded and replaced with a new tip before continuing to add the solution. The tips of dropper bottles should never touch the tube itself. When using reservoir troughs, each reagent should have its own separate tray and the tray should be labeled with the name of that reagent. Using the same tray for all reagents is to be avoided

When adding samples/controls to wells, it is best to work in order from negative controls/samples, to unknowns, followed lastly by highly suspect or positive samples/controls. This will help avoid contamination of negative samples with those that are positive.

4. Washing

Washing is the most critical step in running an ELISA. During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to gently swirl and fill each well. Each well should be washed at least three times in between steps. The wash solution must be diluted as per instructions in the protocol. Be sure to add the proper amount of Tween to each batch of wash solution. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.


Go to Troubleshooting
 

Top
Dot Blot Test Format - Tips
1. When adding samples to the membrane, avoid adding pieces of tissue

Tissue debris that is baked onto the membrane can make it more difficult to interpret results at the end of the test. These extra pieces of tissue tend to trap the development solution causing non-specific color development. After processing, boiling and cooling the samples, only use the supernatant as the sample source. If tissue debris is unavoidable, make notes on the membrane-tracking chart showing where the tissue is located, size, and color. This will help it to be distinguishable from a true signal at the end of the assay.

2. After the membrane has been spotted and baked, note any distinguishing marks on the tracking chart provided

Viewing the membrane before beginning the test will greatly help with interpreting the end results. Any tissue debris, tears, punctures, or deep creases in the membrane can trap the development solution causing non-specific color to be seen. Making notes of these occurrences can assist in determining whether a color change is cause by a specific reaction or if it is due only to trapping of the development solution. Supernatant from processed tissue and hemolymph can also leave brown, pink, or green staining on the membrane after it is baked. This is usually washed away during the procedure however, it is still important to make note of these marks in case there is any difficulty interpreting a true positive reaction.

3. Avoid touching the membrane

The nylon membrane is easily damaged if handled incorrectly. The membrane should never be handled with bare hands. Gloves or forceps should be used at all times. The gloves must be powder free, as the powder will interfere with the membranes' ability to absorb solutions. When it is necessary to pick up or move the membrane, handle it only by the edges, being careful not to touch areas that contain sample. Leave the protective paper on the membrane when it is not being used. This will prevent any damage to the surface of the membrane.

4. Preparing membranes for incubation

During testing it is best to position the membranes as close to the bottom of the bag as possible. This will allow the top of the bag to be folded down during incubation steps causing the solution within the bag to thoroughly cover the membrane during the entire incubation period. If there are multiple membranes within the same bag it is also important that they are not allowed to overlap. Overlapping areas may be blocked from receiving solutions. Bubbles in the solutions can also block solutions from reaching the membrane. Removal of any bubbles is recommended. If they cannot be completely removed, they should not be allowed to sit on top of the membrane. To best remove bubbles, lay the bag flat, then gently push the bubbles toward the top of the bag away from the membrane. Also, tilting the bag slightly will help.

5. Pay close attention to temperature stated in the protocol

It is extremely important that each temperature stated in the protocol is reached and maintained for the duration of incubation. When boiling the samples and the hybridization solutions, the tubes must be placed directly into the boiling water. Be sure each tube is capped tightly to keep water from splashing into the tube. After the 10 minute incubation, the tubes need to be placed into an ice bath to rapidly decrease the temperature to 4°C. This rapid cooling will prevent the separated strands of DNA from reannealing. The same is true for the samples. They must be placed on ice for at least 10 minutes before use and must remain on ice until use.

All reagents should be warmed to room temperature before use unless another temperature is stated in the protocol. The reagent should be kept out of direct sunlight and should be warm to the touch before use. If a reagent is to be used at another temperature, it must be warmed/cooled to the proper temperature before being added.

During the hybridization step, the incubator being used must be maintained at a constant and correct temperature as stated in the protocol. Place a thermometer next to the bags to ensure that the temperature in the area where the bags are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the entire three hours as stated in the protocol.

6. Adding and removing solutions

When dispensing solutions into the bags, be careful not to touch the bag with the tip of the pipette. If the tip does touch the inside of the bag, it must be cleaned thoroughly before being used again or discarded. Contamination of the stock solution or other bags can occur if this is not done. Care should also be taken not to dispense too much air into the bags, as this will cause bubbles that will need to be removed.

When draining or dumping the solution from the bags, it is important to remove as much of each solution as possible. After draining the solution, lay the bag flat and gently push the remaining solution from the bottom of the bag up to and out of the top of the bag.

7. Interpreting the membrane

Each membrane should be interpreted after the 10 minute incubation in stop buffer. As the membranes continue to incubate in the stop buffer, color development may slowly continue causing false positive results. If left overnight in the buffer, it will be hard to distinguish true positive results from false positive color changes. It is best to read the membranes while they are still wet. When the membrane is ready to be interpreted, remove it from the bag and note any color changes on the tracking chart. Once the membrane dries, weak positive results may fade so it is important that notes be made on the tracking chart for accurate record keeping.


Go to Troubleshooting
 

Top
In Situ/Immunohistochemistry - Tips
1. Check to be sure all wax has been removed before beginning the test
The first 65°C incubation period of the test is to assist in melting the wax, making it easier for the xylene or xylene substitute to completely remove it. After the 30 minutes incubation, view the slides and check to see that the wax has melted. If there are still solid, white areas of wax on the slide, place it back at 65°C and check every 10 minutes until all wax has melted. Each slide should be then placed directly into xylene or xylene substitute. Do not allow slides to cool in between taking them out of the incubator and placing them in the xylene. As they cool the wax will begin to harden. If the xylene or xylene substitute is being reused, and there is a problem getting all of the wax off of the slides, the xylene should be discarded and replaced with fresh solution.

2. Adding solutions to the slides
Whether pouring solutions into a container that is holding the slides, or dispensing the solutions directly on to the slide, it is best not to pour/pipette the solutions directly on top of the tissue itself. Direct addition of solutions can disrupt the tissue and possibly wash the tissue off the slide if the pressure is too hard. If slides are in a jar or tray, pour the solution down the slide of the jar/tray and allow it to slowly cover the slide. When pipetting solutions onto the slide, dispense drops on the sides of the tissue and allow the solution to flow over the tissue. Taking these extra precautions will result in better tissue integrity at the end of the test.

3. Pay close attention to temperatures stated in the protocol
It is extremely important that each temperature stated in the protocol is reached and maintained for the duration of incubation. When boiling the hybridization solution, the tubes must be placed directly into the boiling water. Be sure each tube is capped tightly to keep water from splashing into the tube. After the 10 minute incubation, the tubes need to be placed into an ice bath to rapidly decrease the temperature to 4°C. This rapid cooling will prevent the separated strands of DNA from reannealing.

All reagents should be warmed to room temperature before use unless another temperature is stated in the protocol. The reagent should be kept out of direct sunlight and should be warm to the touch before use. If a reagent is to be used at another temperature it must be warmed, cooled to the proper temperature before being added.

During the duration of the hybridization step the incubator being used must be at and remain at the correct temperature as stated in the protocol. Place a thermometer next to the slides to ensure that the temperature in the area where the slides are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the incubation period as stated in the protocol.

4. Adding the hybridization solution (In Situ only)
The hybridization solution must be boiled then placed on ice for at least 10 minutes before it is ready for use. The slides should then be taken out of the paraformaldehyde solution and placed at 95ºC. This can be done using a heat block, oven set to 95ºC, or by suspending the slides over boiling water (the slides should be as close as possible to the surface of the water without allowing water to splash over the slides). Next, add the hybridization solution followed by a coverslip. It is important to add the coverslip immediately after adding the solution to avoid evaporation of the solution. If a number of slides are being run at the same time, add the solution then a coverslip to each slide before continuing on to the next slide.

5. Removing the coverslip
Care should be taken when removing the coverslip so that the tissue is not torn. Tilt the slide to the side and gently shake it. The coverslip should easily fall off. If there is not enough solution on the slide to allow the cover slip to slide off, flood the slide with 1XTBS Buffer then tilt the slide and remove the coverslip.

6. Dehydrate and seal the slides for long term storage
If slides are not going to be interpreted within a short time after being run, or if they are going to be cataloged for future use, it is recommended that they be dehydrated to preserve the tissue. After dehydration, a non-aqueous mounting solution should be used. Once the mounting solution has dried (usually several hours to overnight), clear nail polish can then be used to seal the edges of the coverslip preventing air from getting under the coverslip causing air pockets or further dehydration.


ELISA for lab or field - Troubleshooting
(Enzyme Linked Immunosorbant Assay)
 
High Background

Possible cause: Insufficient washing, improperly made wash solution, or old wash solution.

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to gently swirl and fill each well. Each well should be washed at least three times between steps. As a rule, the plate can never be washed too much only too hard. After the last wash has been completed, any solution remaining in the plate should be further removed by banging the plate against a cloth or paper towel. The wash solution must be diluted as per instructions in the protocol. Be sure to add the proper amount of Tween to each batch of wash solution. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Substrate is not fresh.

Solution: Add the hydrogen peroxide to the ABTS solution just before it is to be added to the wells. After the addition of hydrogen peroxide, the solution must be used within 30 minutes.

Possible cause: Partial contamination of the substrate with the conjugate.

Solution: Change pipette tips between the additions of each reagent. If using reservoir trays, separate trays should be designated for each reagent. Carry over from using the same tray for each reagent can contribute to contamination. If the bottle of ABTS becomes contaminated it will turn a dark green similar to the color of the high positive control or darker. Be aware that the solution does have a green tint to it. This is ok for use.

(To test whether the ABTS is still good, using a clean test tube, add 100 to 200µl of ABTS and 1µl of hydrogen peroxide. Shake well then add 10µl of Streptavidin HRP. Color development should occur within a few minutes.)

 

Rogue wells/ Variation between duplicates:

Possible cause: Improper washing

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to gently swirl and fill each well. Each well should be washed at least three times between steps. As a rule, the plate can never be washed too much only too hard. After the last wash has been completed, any solution remaining in the plate should be further removed by banging the plate against a cloth or paper towel. The wash solution must be diluted as per instructions in the protocol. Be sure to add the proper amount of Tween to each batch of wash solution. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Pipetting errors.

Solution: Each well should receive the same amount of sample/reagent. Any variation in volumes from well to well can lead to variations in signal intensity at the end. See section titled "Tips" for more information on proper pipetting techniques and the proper way to dispense solutions from a dropper bottle.

 

All wells including negative controls turned green/blue

Possible cause: Contamination of the substrate with the conjugate.

Solution: Change pipette tips between the additions of each reagent. If using reservoir trays, separate trays should be designated for each reagent. Carry over from using the same tray for each reagent can contribute to contamination. If the bottle of ABTS becomes contaminated it will turn a dark green similar to the color of the high positive control or darker). Be aware that the solution does have a green tint to it. This is ok for use.

Possible cause: Contamination between samples or controls.

Solution: Always change pipette tips in between samples, controls and all other reagents. It is best to work in order starting from known negatives, to unknown samples and ending with known or high suspect positive samples. When preparing samples for testing, change gloves between samples. If this is not possible, wash hands or gloves thoroughly between samples.

Possible cause: Substrate is not fresh.

Solution: Add the hydrogen peroxide to the ABTS solution just before it is to be added to the wells. After the addition of hydrogen peroxide the solution must be used with in 30 minutes.

No Color. Even controls did not produce a signal

Possible cause: Reagents were forgotten or added in the wrong order.

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times were not followed.

Solution: Be sure to pay close attention to times set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.

Possible cause: : If using ABTS, hydrogen peroxide may not have been added.

The hydrogen peroxide is necessary in order for the substrate and the conjugate to react. Be sure this is added to the ABTS before use.

Unexpected low readings

Possible cause: Washing was done too hard.

Solution: The wash solution should be allowed to swirl gently into each well. The stream of wash solution should be hard enough to wash away any bubbles or debris that may be in the wells but not so hard that it will strip away the antibody coated onto the plate or disrupt any specifically bound reagents.

Possible cause: Reagents not warmed to room temperature before use.

 

Solution: All reagents must be at room temperature before use. Remove the solutions from the refrigerator ahead of time and place in an area away from direct sunlight. Each solution should be warm to the touch before use.

Possible cause: Wrong filter used to take readings.

Solution: Check to see if the proper filter is being used. Each substrate will have a different absorbance reading. Refer to the insert for correct filter information.

Unexpected high readings

Possible cause: Insufficient washing.

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to swirl and fill each well. Each well should be washed at least three times in between steps. The wash solution must be diluted as per instructions in the protocol. Be sure to add Tween to each batch. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Wrong filter used to take readings.

Solution: : Check to see if the proper filter is being used. Each substrate will have a different absorbance reading. Refer to the insert for correct filter information.


Go to Tips
 

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Dot Blot - Troubleshooting
High Background

Possible cause: Each solution was not completely removed before subsequent reagents were added.

Solution: Remove as much of the solution as possible before addition of the next reagent. This is especially important after the addition of the Anti-Dig/AP conjugate. The development solution reacts with the AP in the conjugate. The AP will react whether it is specifically bound to the DNA probe or remaining in the bag due to insufficient washing. Washing steps are included in the protocol to assist in removing non-reacted reagents.

Possible cause: Membranes were allowed to dry out at some point during testing.

Solution: Be sure that each solution covers the entire membrane for the duration of each incubation step. The membrane should not be allowed to dry out between steps. To ensure that the membrane will remain covered by the solutions, after removing bubbles, fold the bag down until it is just above the edge of the membrane.

Light circles or fuzzy areas on the membrane

Possible cause: Light circles can be caused by bubbles in the solution remaining in the bag during incubation periods.

Solution: Remove all bubbles from the bag before beginning incubations. If all bubbles cannot be removed, be sure that they do not sit on top of the membrane. This can be done by laying the bag flat and gently pushing the bubbles towards the top of the bag and away from the membrane.

Possible cause: Mishandling of the membrane either by touching the membrane with bare hands, using gloves containing powder, or folding the membrane

Solution: Never touch the membrane with bare hands. Always use powder-free gloves. Keep the protective paper on the membrane until use. When handling the membrane, it is best to hold it by the edges only using gloved hands or clean forceps.

Possible cause: Membranes were allowed to overlap during testing.

Solution: Do not allow the membranes to overlap during any incubation step. This is especially important (with the membranes in the bag that receives hybridization solution) during the hybridization and development steps. Overlapping of the membranes will prohibit solutions from reaching all sections of each membrane.

No positive signal seen even with control membranes

Possible cause: Reagents not warmed to room temperature before use (unless otherwise stated in the protocol).

Solution: Remove reagents from refrigerator and allow them to warm to room temperature completely before they are used. Any reagents that require further heating need to be at their required temperature before use.

Possible cause: Insufficient denaturation of the probe.

Solution: The probe must be placed directly in boiling water for 10 minutes. A reduction in time or temperature may inhibit the two strands of the probe from completely separating.

Possible cause: Insufficient cooling of the probe.

Solution: After boiling the probe it must be immediately placed at 4°C for 10 minutes before it can be added to the membrane. This drastic cooling process keeps the two separated strands of the probe from coming back together until they can be added to the membrane.

Possible cause: Incorrect hybridization temperature or time.

Solution: The incubation temperature specified in each protocol must be maintained for the duration of the hybridization step. Place a thermometer next to the bags to ensure that the temperature in the area where the bags are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the entire three hours as stated in the protocol.

Possible cause: One or more of the reagents were not added or used in the incorrect order.

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times and temperature were not followed.

Solution: Be sure to pay close attention to times and temperatures set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.


Go to Tips
 

Top
In Situ/Immunohistochemistry - Troubleshooting
Poor tissue quality
Problem: Tissue sections fall off slide during testing.

Solution: Positively charged slides must be used when preparing slides (see below). During the Proteinase K step, do not allow the incubation period to exceed the time noted in the protocol. Allowing the Proteinase K to stay on the slides too long can cause the tissue to be degraded and fall off. The tissue is very vulnerable during and just after this step so care should be taken when adding and removing reagents and/or cover slips.

Problem: Tissue sections are badly disrupted at the end of the test.

Solution: The entire in situ procedure is hard on the tissue. Proteins that hold the sections together are being weakened. Special care and consideration should be taken thorough out the procedure. See the section titled " Tips" for more information on how to properly add reagents, wash slides and remove cover slips with limited distress to the tissue.

Too much background or nonspecific staining
Possible cause: Tissue was badly disrupted leading to many areas were the development solution can be trapped.

Solution: Care should be taken throughout the process to avoid excess tissue damage.

Note: Nonspecific staining can be easily distinguished by it having no cellular morphology. Cuticular staining and areas where the reagents were nonspecifically trapped during testing, will usually have a uniform appearance and will not show any cell association or morphology.


Possible cause: Tissue sections were allowed to dry out at some point during testing.

Solution: Once the test has begun, the tissue section should never be allowed to dry out. Each solution must cover, and remain covering the entire tissue section during the entire incubation period.

Possible cause: Development solution remained on the slides in excess of two hours or development was carried out in the light.

Solution: The development solution should be removed and inactivated after two hours. During incubation, the slides should be placed in a humid chamber and covered or placed in a dark area away from all light.

No staining seen even on positive control slides
Possible cause: Reagents not warmed to room temperature before use. (unless otherwise stated in the protocol).

Solution: Remove reagents from refrigerator and allow them to warm to room temperature completely before they are used. Any reagents that require further heating need to be at their required temperature before use.

Possible cause: Insufficient denaturation of the probe.

Solution: The probe must be placed directly in boiling water for 10 minutes. A reduction in time or temperature may inhibit the two strands of the probe from completely separating.

Possible cause: Insufficient cooling of the probe.

Solution: After boiling the probe it must be immediately placed at 4°C for 10 minutes before it can be added to the slides. This drastic cooling process keeps the two separated strands of the probe from coming back together until they can be added to the slides.

Possible cause: Incorrect hybridization temperature or time.

Solution: The incubation temperature specified in each protocol must be maintained for the duration of the hybridization step. Place a thermometer next to the slides to ensure that the temperature in the area where the slides are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the duration of hybridization as stated in the protocol.

Possible cause: One or more of the reagents were not added or used in the incorrect order

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times and temperature were not followed.

Solution: Be sure to pay close attention to times and temperatures set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.


Possible cause: Insufficient removal of the wax during rehydration

Solution: Each slide should be viewed after the 65°C melting step to be sure that the wax appears melted. As quickly as possible the slides should be taken from the incubator and placed directly into xylene or xylene substitute. If the xylene or xylene substitute is being reused, and there is a problem getting all of the wax off of the slides, the xylene should be discarded and replaced with fresh solution.

Possible cause: Proteinase K is not active.

Solution: Repeated freezing and thawing of the Proteinase K solution will result in loss of activity. If the entire solution is not being used, the remaining solution should be divided into smaller volumes and frozen. When ready to use, remove only the amount needed from the freezer and thaw on ice. Keep the solution on ice until it is ready to be added to the slides. Proteinase K is used to weaken tissues and cell walls, allowing the reagents access to target DNA.