[Histonet] ChAT staining of nerves / spinal cord

[Melanie Walker]

Hello Histonet,
I hope you can help me with some troubleshooting. I am having some background issues that I am not sure how to get rid of. I actually am working with two different ChAT antibodies and trying to get at least one of them up and running.

I am performing IHC on FFPE Sheep sciatic and spinal cord. I am trying to develop either a Goat polyclonal ChAT or Rabbit Polyclonal ChAT.

I started with the goat anti chat  but I was getting tons of background due to the fact that my secondary is rabbit anti goat and is binding all over my sheep tissue due to the similarities between the species (literally everything was stained equally - it was impossible to tell if there is any positive staining of the ChAT b/c so much background). So, I have since ordered a rabbit anti-sheep Fc frags for blocking the sheep tissue before applying the primary. This did provide some blocking capabilities (negative on the nerve tissue but still have pretty intense background staining of collagen tissue, particularly in the dura around the sc and the nerve sheaths). Also, I can now see that we do not have specific binding of the ChAT antigen.

Most recent Protocol for Goat anti Chat antibody: ** double rinse w/ pbs tween and blow between each.
1. Run stain at 1:50, 1:100, 1:250, negative (diluent only)
2. Heat retrieval in pressure cooker for 5 min (set#1) and no heat retrieval (set #2)
3. Apply Background sniper**
4. Apply Fc frags anti-sheep**
5. Apply primary for 1 hour**
6. apply goat probe 15 min**
7. Apply goat polymer 15 min**
8. DAB 5 min* rinse, no blow.


Due to all of the trouble I was having with the homologous tissues of goat and sheep I decided to try a rabbit polyclonal ChAT and thought everything would work out super peachy... however I have had some troubling results. My biggest concern is that we were getting appropriate staining on the very first run w/ only light background at 1:250, however when I tried to repeat did not get any staining!!! I wonder if a short heat retrieval with a good solution such as DIVA(which works for me for some of my more difficult antibodies) would be any use. Also, I wonder if doing a cold 4C/long 12 hr primary incubation would be helpful.

ChAT Rabbit Polyclonal
11/21/11

1:50, 1:100, 1:250

No Heat Retrieval

Primary Antibody
1 hour

1:50 = Very Dark w Background.
1:100 = Unacceptable Background
1:250 = Light Background with Appropriate staining of Nerve cells.
Negative slide was clean.

ChAT Rabbit Polyclonal
11/28/11

1:250, 1:400

No Heat Retrieval

Primary Antibody
1 hour

1:250 = Light background no staining of nerve cells.
1:400 = Very light background no staining of nerve cells.
Negative slide was clean

ChAT Rabbit Polyclonal
11/28/11

1:250, 1:400

With Heat Retrieval
Citrate Buffer pH = 6 for
15 minutes

Primary Antibody
1 hour

1:250 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
1:400 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
Negative slide = ? cannot locate right now?

ChAT
Rabbit Polyclonal
12/7/11

1:100
1:250

Heat Retrieval
Citrate Buffer pH = 6 for
5 minutes

Primary Antibody
3 hours

1:100 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
1:250 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
Neg = clean

ChAT
Rabbit Polyclonal
12/7/11

1:100
1:250

No Heat Retrieval

Primary Antibody
3 hours

1:100 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
1:250 = Dark background, dark staining of nerve cells but could not be differentiated from background staining.
Neg =Clean

If you have ANY advice on what route should be taken at this point I would love to at least have a plan of attack for my next test runs.
If you have any questions that may help you give me some advice I would be happy to share :)

Thank you!!

Melanie