I am working on apoptosis. I have done PCR and Western Blot for my studies, and finally want to confirm by immunohistochemistry.
If you need the simplest IHC protocol, we practice the following.
A. Deparaffinize section and rehydrate as follows:
1. Place slides in a preheated incubator at 70C for 30 min.
2. Two times for 5 minutes in xylene
3. 100% ethanol for 5 minutes
4. 95% ethanol for 5 minutes
5. 80% ethanol for 5 minutes
6. 70% ethanol for 5 minutes
7. Distilled Water for 5 minutes
B. Antigen retrieval
1. Arrange slides in a Coplin jar with antigen retrieval solution of choice (10mM citrate, pH 6/ 10mM EDTA pH 8 ).
2. Place Coplin jar in microwave or water bath or oven at required temperature..
3. Remove Coplin jar from microwave or water bath or oven and allow to cool for 20 minutes at room temperature.
4. Rinse sections in PBS , two times for 5 minutes.
5. Cover the slide with endogenous oxidation blocking solution for 10 min.
6. Rinse slides for 5 minutes in PBS.
C. Staining
1. Incubate sections in primary antibody for ½ to 1 hour at room temperature in a humid chamber to prevent air drying of the tissue sections.
2. Rinse sections twice for 5 minutes in PBS.
3. Gently remove excess PBS and cover sections with secondary antibody for 30 minutes at room temperature in the humid chamber.
4. Rinse sections twice for 5 minutes in PBS.
D. Chromogen and counter stain
1. Incubate sections for approximately 2 minutes in DAB (1 drop diaminobenzidine + 1 ml peroxidase substrate solution or 20 ul diaminobenzidine in 1ml peroxide substrate solution).
2. Rinse slides well three times for 10 minutes in PBS.
3. Counter stain with hematoxylin (2-3 dips).
4. Rinse slides three times for 5 minutes with PBS.
5. Dehydrate two times for 2 minutes in 95% ethanol; two times for 2 minutes in 100% ethanol and two times for 2 minutes in xylene.
6. Mount slides with DPX (contains; xylene & Dibutyl phthalate) .
You can find good tips and different options as per your requirement on http://www.ihcworld.com/protocol_database.htm or abcam also.
Best of luck.
Most Bcl2 antibodies- or better all I have seen- work very very poorly on FFPE (formalin-fixed, paraffin-embedded) tissues. It is highly preferable to use frozen sections. Lymph nodes are nice positive controls. Bcl2 is highly expressed in them and even FFPE lymph nodes should give you a kind of positive result. But nothing compared to stainings of frozen sections!
For frozen sections, cold acetone treatment ("fixation") should be fine.
Again, like many other questions here, the question itself is very vague: have you tried anything already. If yes, what did you try. What controls did you use, what antibodies etc. The more info you give, the better the answers will be...
- Badges
- Science method