General Protocol - Immunocytochemistry
Prepare paraffin embedded sections
1. Cut small blocks of tissue (1 cm2 x 0.4 cm) and place them in freshly prepared 4% paraformaldehyde.
2. Incubate for 2 h to overnight.
3. Embed the tissue in paraffin using standard methods.
4. Cut sections of 2-5 µm (thinner sections will give better results).
5. Float the sections on warm (not hot) distilled water. Pick up on poly-L-lysine- or silane-coated microscope slides.
6. Dry the sections overnight at 37°C.
7. Remove the paraffin in 2 changes of xylene, 3 min each. Take through graded alcohols to distilled water (twice 3 min of absolute ethanol, followed by 2 changes of 95% ethanol, 3 min each, and a final rinse in distilled water).
8. Perform protease and/or heat-mediated antigen retrieval as required using standard methods.
9. Place slides in buffer (PBS or TBS).
10. Proceed with immunostaining as described below.
Prepare fresh frozen sections
1. Cut fresh sections of 5-10 µm from snap-frozen block of tissue and pick them up on poly-L-lysine- or silane-coated microscope slides.
2. Depending on the antigen and tissue, different steps can be used next:
a) Air-dry the slides overnight at room temperature, or
b) Fix the section in 100% acetone for 30 sec at room temperature, or
c) Air-dry the section and dip in paraformaldehyde for 2 min. Wash with several changes of PBS and place in 1% NP40/PBS for 5 min.
3. Rinse the section in several changes of PBS.
4. Place slides in PBS.
5. Proceed with immunostaining as described below.
Prepare whole-cell preparations
1. Wash the fluid off the cells with buffer (PBS or TBS). Cells can be monolayers, cytospins or cells allowed to settle on coated slides from suspension.
2. Depending on the antigen and tissue, different fixation methods can be used. In general, paraformaldehyde or glutaraldehyde are used for peptides and smaller proteins, while acetone or methanol is used for large proteins.
a) Fix the cells in 100% methanol, 100% acetone or 50% acetone/50% methanol. Incubate for 2 min at room temperature with gentle agitation, or
b) Incubate the cells in 4% paraformaldehyde for 10 min at room temperature or in 1% glutaraldehyde/PBS in a fume hood for 1 h at room temperature. Wash the slides twice with buffer (PBS or TBS). If intracellular antigens are to be detected, it may be necessary to permeabilize the cells by soaking them in buffer (PBS or TBS) with detergent (0.2% Triton X-100 or 0.05-2% Tween 20 or 0.1% Saponin) for 30 min.
3. Rinse slides in buffer (PBS or TBS) with 4 changes over 5 min.
4. Proceed with immunostaining as described below.
Immunostaining
1. Take the slide from the buffer and shake off excess buffer. Dry the slide with a paper tissue, except for the section area.
2. Place the slide(s) on a rack (made of wooden sticks or glass rods) in a Petri dish with some wet (distilled water) paper tissue or cotton wool to keep the atmosphere humid.
3. Dilute the primary antibody at its optimal concentration (usually 1-10 µg/ml for monoclonals) in 3%BSA/PBS or 3%BSA/TBS. Cover the section with the diluted antibody (average is 100 µ l/section). Cover the dish and incubate for 1 h at room temperature or overnight at 4°C.
4. Drain the slide briefly on to a paper tissue. Wash 3 times with buffer (PBS or TBS) for 5 min.
5. Dry the slide as in step 1. Dilute the secondary antibody at its optimal concentration in 3%BSA/PBS or 3%BSA/TBS and apply to the section. Cover the dish and incubate for at least 30 min at room temperature.
6. Wash as in step 5.
7. Perform detection using standard methods.
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