Hyde Lab Protocols: Immunohistochemistry on Cryosections

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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