Hi Pratik,

If budget is not an issue, you can use the kits available from DAKO. Vector labs also have good kits with large number of citations for each. The IHC steps to follow will be determined by fixation/preservation method. For example, formaldehyde-fixed parafilm-embedd (FFPE) sections have to be first deparafinized with xylene and rehydrated with ethanol (start with 100%, 95%, 80%, 70%, 50%, then rinse in PBS). This is followed by antigen retrieval at either high or low pH, depending on the antigen for the the antibody was recommended, at 95 degrees Celcius for 10 min (some protocols recommended longer time), followed by cooling at room temperature for 30 min.

I used the following steps when I used the DAKO LSAB kit for FFPE sections of lymph nodes:

1. Deparanize as described above.

2. Rehydrate in ethanol as described followed by one or two rinses in PBS

3. Antigen retrieval at high or low pH (as described above).

4. Block 3% hydrogen peroxide for 10 min at room temperature.

5. Serum blocking is optional.

6. Incubate sections in primary antibody (dilution may vary from assay to assay) either for 1-2 hr at RT or overnight at 4 degrees. You will need to use a hydrophobic pen to make a dam around the section to prevent the diluted antibody from spreading away from the tissue.

7. Wash 3X in either PBS or the recommended wash buffer.

8. Incubate in Biotinylated anti-rabbit and anti-mouse immunoglobulins (I used a mouse monoclonal primary antibody) for 10 min or as recommended. Add enough Biotinylated anti-rabbit or anti-mouse immunoglobulins to cover tissue.

9. Rinse briefly in PBS (or recommended wash buffer), wipe off excess PBS and incubate in streptavidin-conjugated peroxidase for 10 min or as recommended. Add enough streptavidin-HRP to cover the section.

10. Rinse as before.

11. Develop with chromagen (away from light if possible). For this kit, I diluted 1 drop of chromagen in 1 mL PBS. I usually monitor this step under the microscope to determine level of staining to avoid over staining which can produce high background. Wash sections immediately in running tap water.

12. Counterstain in hematoxylin. I usually do this for 22 to 30 seconds in hematoxylin from Vector lab, to avoid over staining. Then immediately washed with running water.

13. Dehydrate the sections in ethanol (50%, 705, 80%, 95%, and 100 %) then xylene, dry, and mount with VectaMount permanent mounting medium (from Vector lab).

Good luck.

Can I use them for western blot analysis?

Dear Pratik,

As Sebastian said above that you need someone's (may be core facility in your University or watch some you tube videos) help for basic steps and tricks to get on Immunohistochemistry. Apparently it is not that easy if you go as an inexperienced person.

Still you should be able to rescue these tissues (but next time you should freeze them in OCT blocks after fixing first, read my enclosed papers). For now you need to place them in mould containing OCT so that you can cut sections of frozen blocks. You may like to do like this as below:

For these tissues, you need to buy plastic mould of right size and OCT. Place your empty mould on a horizontal piece of dry ice, let the mould cool down for 5 minutes, place your frozen tissue ( from -80C freezer) in the middle of mould (while on dry ice) and fill the mould with OCT. In this way your tissue sample will be fully covered in OCT and that will make frozen block, place the frozen block at -80C. Next day or after one hour, you can cut sections from this frozen block by cryostat machine (thickness of 7um to 10um). Once sections are collected, place them on slides and place slide in a slide box kept at dry ice in a styrofoam box. Once all sections are collected, place slide box at -80C.

Take out slides from -80C box and bring slides (for IHC for that assay) to room temperature for 15-20-30 minutes till sections settles to slide surface, wash once with few drops of cold (on ice or 4C) 4%PFA and than place few drops of fixative 4%PFA on the sections and place the slides at 4C for 30min, wash the fixatiive 2 times with PBS, place a drop of 0.2% glutaraldehyde at 4C (freshly diluted) for 5 min. Wash with PBS-T 2 times and Proceed with immunohistochemistry. if you have any question, e-mail me at [email protected] or address here at RG. (when using fixative steps, use slides in a close box preferably so that its vapors do not harm people around)

You can certainly use for WB.