6.8 Detailed Protocol for Conjugate IF-EM AT Using
Freeze Substitution and Embedding in Lowicryl
HM-20
1. Tissue Fixation: Chemical fixation by perfusion
1A. Required materials and equipment:
Anesthetic
Paraformaldehyde (reagent grade, crystalline; Sigma-Aldrich #P6148)
Glutaraldehyde (50%, EM grade; Electron Microscopy Sciences #16310)
Sodium phosphate, monobasic, monohydrate
Sodium phosphate, dibasic, anhydrous (or dibasic heptahydrate)
Sodium chloride
Sodium heparin
Hazardous waste collection containers
Fume hood
Surgical tools
Gloves, safety glasses, coats (PPE)
Storage vials or tubes (20 ml scintillation vials or culture/centrifuge tubes)
1B. Preparations:
0.1M phosphate buffer, pH 6.8 (PB 6.8):
6.93 g sodium phosphate, monobasic, monohydrate
7.06 g sodium phosphate, dibasic, anhydrous (or 13.32 g dibasic heptahydrate)
Distilled or deionized water up to 1 L, titrate as needed
Fixative: 2% paraformaldehyde/2% glutaraldehyde in PB 6.8. Prepare same
day, keep at room temperature,
For 100 ml (per mouse):
2 g paraformaldehyde
Dissolve in warm, but not boiling:
100 ml PB 6.8
Cool solution to room temperature, add:
4 ml 50% glutaraldehyde
Filter (we use vacuum filtration with Whatman #40 filter paper)
Heparinized normal saline
9 g sodium chloride
1 L distilled or deionized water
Add 1000 units/100 ml of sodium heparin
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K. D. Micheva and K. D. Phend