High Performance Immunocytochemistry

Basic Working Reagents

• Prepare a 100X dilution of a selected Series 100 Antiserum in 1% GSPBT
• PB = 0.1 M phosphate buffer, pH 7.4
• PBT = PB + 0.05% thimerosal, pH 7.4
• 1% GSPBT = 1% goat serum in PBT
• 2° Ab = 1 nm Gold conjugated or fluorophore conjugated anti-rabbit IgG
• Mounting medium

Silver intensification solutions

• Stock A = 114 mg citric acid + 342 mg sodium citrate in 6 ml deionized water (make fresh - do not store)
• Stock B = 0.5 g hydroquinone in 15 ml deionized water (make fresh - do not store)
• Stock C = 1% aqueous silver nitrate (may be stored indefinitely at room temperature wrapped in foil)
• Working solution = 5 ml A + 1 ml B + 1 ml C, in that order, made up immediately before use
• Stop solution = 5% acetic acid
• Optional: Slide warmer set at 30°C (Avoid cold surfaces. Temperatures below 24°C slow the reaction).

Procedure

1. Sections of epoxy resin embedded tissue (100-1000 nm thick) on spot slides.
2. Deplasticize (see Methods for protocol).
3. Deosmicate if necessary (see Resources for protocol)
4. Probe with Signature Immunologics antiserum 4 hours to overnight at 25 microliters/well. Cover with an inverted glass staining dish sealed with plastic wrap to prevent evaporation.
5. Flick off antiserum. Dip once in 0.1 M PB to rinse off excess and wash 10 minutes in 1% GSPBT; use plastic mailing cassettes that hold 5 slides (requires about 15 ml of solution).
6. Dip slides in deionized water and dry with an air canister. Incubate 60 min with 25 microliters/well of 2° Ab in 1% GSPBT (e.g. a 1 nm gold goat anti-rabbit IgG or a fluorophore conjugated anti-rabbit IgG). Silver-intensification methods are the most sensitive and are archival.
7. Flick off 2° Ab. Dip in 0.1 M PB to rinse off excess. Wash in PB 1 hour in plastic cassettes.
8. Dip in deionized water and dry with an air canister. Mount in a medium of your choice if using fluorescence. Otherwise, go to the next step.
9. Prepare silver intensification solutions. Stock solution C may be prepared in advance. Prepare solutions A and B prior to intensification and use within 1 hour.
10. Use the working solution immediately as it lasts only 10 min. Expose sections to the solution for 4-8 minutes in a dark location, such as under an aluminum cover. You needn't work in absolute darkness, but the background decreases slightly if the intensification occurs away from bright light. The reaction is temperature sensitive and slows substantially below 24°C.
11. Stop the reaction with a brief dip in 5% acetic acid.
12. Wash for 10 minutes in deionized water and dry with an air canister.
13. Cover slip in a medium of your choice.