Enhanced HPI^tm

Protocol for Zero-Low Glutaraldehyde / High Paraformaldehyde Fixation

Series 100 IgGs can be used with low glutaraldehyde levels provided enhanced visualization is employed to detect IgG binding. Reduction in glutaraldehyde levels will cause substantial loss of endogenous amino acids from tissues. Formaldehyde cross-linking is of low efficiency, leading to diffusion of free or derivatized amino acids out of the sample during processing. However, about 1-5% of the amino acids are retained by methine bridge formation. The following table shows the minimum glutaraldehyde conditions for each IgG when used with 4% paraformaldehyde in 0.1 M phosphate buffer / 3% sucrose fixative for post-embedding immunocytochemistry on thin sections. Performance is also improved with frozen section and tissue culture formats when penetrating reagents such as 0.3% Triton X-100 are employed.

Minimum Glutaraldehyde with 4% Paraformaldehyde

Basic Working Reagents

• Prepare a 100X dilution of a selected Series 100 antiserum in 1% GSPBT or GSPBTX as needed
• PB = 0.1 M phosphate buffer, pH 7.4
• PBT = PB + 0.05% thimerosal, pH 7.4
• PBTX = PB + 0.05% thimerosal, pH 7.4+0.3% Triton-X100
• 1% GSPBT = 1% goat serum in PBT
• 1% GSPBTX = 1% goat serum in PBTX
• 2° Ab = Biotinylated anti-rabbit IgG
• Detection reagent = 1 nm gold conjugated or fluorophore (e.g. Cy3^tm) conjugated streptavidin
• 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 Resources for protocol).
3. Deosmicate if necessary (see Resources for protocol)
4. Start with air-dried slides. Block 60 min in 1% GSPBTX at 25 microliters/well. Flick off droplets and do not rinse. Probe immediately.
5. 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.
6. 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).
7. Dip slides in deionized water and dry with an air canister. Incubate 60 min with 25 microliters/well of biotinylated 2° Ab in 1% GSPBTX.
8. Flick off 2° Ab. Dip in 0.1 M PB to rinse off excess. Wash in 1% GSPBTX 60 min in plastic cassettes.
9. Dip slides in deionized water and dry with an air canister. Incubate 60 min with 25 microliters/well of 1 nm gold conjugated or fluorophore (e.g. Cy3^tm ) conjugated streptavidin.
10. Flick off droplets. Dip in 0.1 M PB to rinse off excess. Wash in 1% GSPBTX 1 hour in plastic cassettes.
11. 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.
12. 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.
13. 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.
14. Stop the reaction with a brief dip in 5% acetic acid.
15. Wash for 10 minutes in deionized water and dry with an air canister.
16. Cover slip in a medium of your choice.