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Leica EM IGL Application Note Mouse pancreas PROTOCOL I: ON-GRID IMMUNOGOLD-LABELING USING SECONDARY ANTIBODIES Mouse pancreas. Cryo fixed followed by freeze substitution and low temperature embedded in HM20. Note: a. This protocol does not cover resin etching for embedded samples and methy lcellulose embedding for cryo-ultrathin sections). b. The whole procedure takes about 6.0 hours. c. If ultrasmall gold conjugates are being used, proceed to Protocol II for silver enhancement after completing Protocol I Wash: PBS, 1 X 2 min. (Slide 1: Wash -PBS) Aldehyde inactivation: 50 mM glycine in PBS, 15 min. (Slide 2: Aldehyde inactivation) Blocking: 0.8% BSA, 0.1% Cold Water Fish Skin gelatin and 5% NS (from the same species that secondary antibody is raised from) in PBS, 15 min. (Slide 3: Bloking) Wash: 0.1% Aurion BSA-c in PBS, 2 X 5 min. (Slide 4-5: Wash-PBS/BSA-c) Primary antibody incubation: dilute antibody with incubation solution and incubate for 2 hour. (Slide 6: Primary antibody) Wash: incubation solution, 6 X 5 min. (Slide 7-12: Wash-PBS/BSAc) Secondary antibody incubation: dilute Aurion colloidal gold conjugated secondary antibodies with incubation solution and incubate for 1.5 hour. For 6-25 nm gold conjugates, use1/20 dilution, and for ultrasmall gold conjugates, 1/100. (Slide 13: Secondary antibody) Wash: incubation solution, 6 X 5 min. (Slide 14-19: Wash -PBS/BSA-c) Wash: PBS, 3 X 5 min. (Slide 20-22: Wash -PBS) Post-fixation: 2.0% Glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), 5 min. (Slide 23: Postfixation) Wash: dd H2O, 3 X 5 min. (Slide 24-26: Wash -water) Unload grids from IGL and wash by dipping grids in a beaker of water before drying sections on filter paper is required.

PROTOCOL II: ON-GRID SILVER ENHANCEMENT Mouse pancreas Cryo fixed followed by freeze substitution and low temperature embedded in HM20. Note: a. This protocol is designed for using Aurion R-gent SE-EM for enhancement of Aurion ultrasmall colloidal gold conjugates. b. This protocol produces particles about 15 nm at 23 °C. But be aware that particles size can vary as enhancement duration, temperature, the batch of reagent changes, and type of sample preparation. c. The whole procedure is about 83 min. Re-hydate sections: PBS, 15 min. (Slide 1: Wash -PBS) Wash: water for embedded sample, and Aurion ECS for cryo-ultrathin sections, 4 X 5 mim. (Slide 2-5: Wash-ECS or Wash-water) Silver enhancement: Aurion R-gent SE-EM (refer to product data sheet for instruction on reagent preparation), 25 min. (Slide 6: Silver enhancement) Wash: water for embedded sample, and Aurion ECS for cryo-ultrathin sections, 4 X 5 mim. (Slide 7-10: Wash-ECS or Wash-water) Wash: water, 3 X 1 min. (Slide 11-13: Wash-water) Unload grids from IGL and wash by dipping grids in a beaker of water before drying sections on filter paper is required.

Immunogold/silver labeling of alphaamylase in Lowicryl HM 20 embedded pancreas tissue using Aurion ultrasmall gold conjugate and R-gent SE-EM silver enhancement reagent. Bar = 2µm Courtesy of Hong Yi, Emory School of Medicine Microscopy Core Laboratory, Atlanta, GA.