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Author Topic: IHC TroubleShooting - Formalin Fixed Bone - OPG Staining  (Read 4067 times)

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Offline JHernandez

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IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« on: September 04, 2015, 10:01:55 AM »
Hello all,

Just looking for some help with my IHC. I am trying to stain OPG in Formalin Fixed Paraffin tissue. Here is the gene info http://www.genetex.com/OPG-antibody-GTX127948.html . I am using Proteinase K retrieval at this concentration:  Working Solution (1x, 20 ug/ml or 0.6 units/ml). Use it at 37C for 15 mins.

We have tried diluting our secondary Ab to 1:1000 to decrease the background. We are noticing that we are able to barely see a couple of cells in the bone marrow that have a localized staining pattern. The only thing is that I am not sure how else to amplify or increase the OPG signal.

I feel that since we fixed our tissue for about 5 days the enzyme method might not be the best way to retrieve. Any suggestions would be appreciated. Thank you.

-Jesse H.  :D

IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« on: September 04, 2015, 10:01:55 AM »

Offline gula

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Re: IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« Reply #1 on: September 04, 2015, 10:55:53 AM »

5 days  fixation is not long for IHC staining.
Why did you choose enzymatic antigen retrieval?
The datasheet doesn't state any antigen retrieval at all at the picture shows tissue stained with 1:500 titer.

I would try to omit the antigen retrieval and see what happens.
gula

Offline JHernandez

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Re: IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« Reply #2 on: September 04, 2015, 11:11:00 AM »
We did receive an IHC protocol from the GeneTex for Heat Retrieval using Citrate Buffer (ph = 6). We decided to try proteinase K because we are still having issues with our tissue sections falling off the slides when using a microwave for heat retrieval. When the heat reaches 100C or more our sections are pretty much toast. For some reason the bone marrow stays on the slides, but the actual bone part of the section comes right off.

-JH

Offline gula

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Re: IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« Reply #3 on: September 05, 2015, 03:03:24 AM »
Heat and proteinase has not the same effect.
There is a relation between time and temperature in HIER. Try a gentle way of HIER without cooking. For example at 80C for one hour.
I assume, that you use adhesive slides and as long as you work with bone, you certainly know the tricks for sections to stay on slides. (like drying over night at 37C, be careful about water under the section)
gula

Re: IHC TroubleShooting - Formalin Fixed Bone - OPG Staining
« Reply #3 on: September 05, 2015, 03:03:24 AM »