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General Protocol for Immunohistochemistry on Cryosections

        Fix two groups of light txt’d eyes: 4% PFA and 9/1 Eth. Form, O/N 4°C.

• 1X PBS wash 20 min

        Infiltrate for TBS embedding

• 5% sucrose/1X PBS 30 min
• 5% sucrose/1X PBS 3 hrs
• 30% sucrose/1X PBS, O/N 4°C
• 30% sucrose/1X PBS:TBS, 1:1, O/N 4°C

        Embed in TBS

• 100% OCT and freeze

         Section

• 12-14 µm 3 PER SLIDE
• Dry on warmer (50°C) 2 hrs
• Alt. step: Can freeze at –80°C to store;before continuing, re-dry on warmer for 20 min.
• Outline with PapPen

        Primary antibody incubation

• Hydrate in 1X PBS in coplin jar, 20 min
• Incubate in blocking solution, 1 hr RT
  
  

Blocking Solution          (10 mL) 2% NGS                      200 µl 0.2% Triton X-100       20 µl 1% DMSO                  100 µl  in 1X PBS                    9680 µl



• Dilute primary antibody in blocking soln
• Incubate in primary antibody soln O/N 4°C

        Secondary antibody incubation

• Wash 10 min in 1X PBS 0.05% Tween-20 (ex. 50 µl Tw-20 in 100 mL 1XPBS)
• Wash 10 min in 1X PBS 0.05% Tween-20
• Wash 10 min in 1X PBS 0.05% Tween-20
• Dilute secondary antibody in 1X PBS 0.05% Tween-20 (1:500 for AF antibodies)
• Incubate in secondary antibody solution 1 hr RT

        Wash off unbound antibody

• Wash 10 min in 1X PBS 0.05% Tween-20
• Wash 10 min in 1X PBS 0.05% Tween-20
• Wash 10 min in 1X PBS 0.05% Tween-20
• Wash 10 min in 1X PBS

        Coverslip

• Use Prolong Gold or Vectashield

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