Immunohistochemistry on whole mount retinas

Immunohistochemistry on whole mount retinas

1) Fix eyeballs for 20min in 4% PFA at RT.

2) Transfer to PBS and dissect right away.

3) Block for 1 hour shaking at RT in TNBT.

4) Incubate with primary antibodies in blocking buffer overnight to 3 days at 4˚C on shaker (staining has to be very strong for light sheet microscropy so longer incubation periods are generally favorable).

5) Wash 1x 5min rocking at room temperatur with PBS.

6) Wash 3x 5min rocking at room temperature with PBLEC.

7) Dilute secondary antibodies (Molecular probes, 1:250) and IsolectinB4 directly conjugated with fluorophore (Molecular Probes, 1:50) in PBLEC and incubate for 2-6 hrs shaking at RT. Wrap plate in foil to avoid light from this step onwards.

8) Wash 3x 5min with PBS.

9) Fix over night in 4% PFA at 4˚C.

10) Wash briefly in PBS.

11) Mount in 2% low melting agarose/PBS for imaging with light sheet. Melt agarose at >65˚C, and then maintain at 42˚C before adding the tissue. Use PBS to fill the chamber when imaging.

Solutions:

TNBT:

100 mM Tris pH 7.4

150 mM NaCl

0.5% Triton X100

0.5% TSA Blocking Buffer Reagent (FP102, Perkin Elmer)

PBLEC:

1mM CaCl2

1mM MgCl2

0.1mM MnCl2

1% TritonX in PBS

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