Immuno-histochemistry – cell & tissue slides
Several protocol variants exist for immuno-histochemistry of cell & tissue slides. The most basic variants are described below.
Chamber slides allow you to grow your cells directly onto the slide for which you will be performing your analyses. Alternatively, cells can be grown on sterile cover slips placed into the wells of normal cell culture plates. When cells are ready for analysis, remove media and wash gently with PBS, fix for 15 minutes with 4% PFA, then wash gently again with PBS. (For more information about PFA see Fixation). Go to step 1 below.
For paraffin sections wash with Xylene for 5 minutes, followed by 100% ethanol for 2 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and finally deionised water for 2 minutes. An optional antigen retrieval step may be performed by boiling for 10 minutes in 10mM sodium citrate pH 6 then cooled for 30 minutes and washed with deionised water. Once the paraffin has been removed a PAP pen may be used to draw a hydrophobic barrier / circle around the specimen on the slide. Go to step 1 below.
For cryo-sections, air dry slides then wash / hydrate slides in deionised water or PBS for 2 minutes. (May need to repeat the 2 minute wash to completely remove the cryo-medium). A PAP pen may be used to draw a hydrophobic barrier / circle around the specimen on the slide. Go to step 1 below.
1. For tissue apply PBST at 4 °C for 5 minutes to permeate membranes. For cells apply PBST for 2 minutes. (PBST = PBS + Triton-X 0.1%).
Important: Do not let slides dry out at any stage. To prevent slides from drying out, slides may be processed in a humidity / moisture chamber.
Important: One slide should be used as a negative control treated in the same way for every step except step 3 where 1% donkey serum or goat serum in PBS should contain no primary antibody.
2. Block slides with 10% donkey serum or goat serum in PBS at room temperature for 1 - 2 hours for tissue or 1 hour for cells. (The species from which the serum is derived should match the secondary antibody species. Serum should not match the species the primary antibody was raised in).
3. Dilute primary antibodies (1 in 200 to 1 in 1000) with 1% donkey serum or goat serum in PBS and apply to slides at 4°C overnight.
4. Wash slides 4 times with PBS for 5 minutes each time.
5. Dilute secondary antibodies (1 in 200 to 1 in 1000) with 1% donkey serum or goat serum in PBS and apply to slides at room temperature for 2 hours. (For fluorescent secondary antibodies incubate in the dark).
6. Wash slides 4 times with PBS for 5 minutes each time.
7. Cover-slip slides with mounting medium and seal cover-slip parameter with nail varnish or similar. Storing at 4°C or in a cool place may increase the life of the signal. For fluorescent secondary antibodies store in the dark. Vectashield mounting medium with DAPI (from Vector Laboratories) is an ideal option. DAPI stains nuclei blue.