Fixed tissue preparation, immunohistochemistry, and imaging

Immunostaining Protocol for ChAT

Blocking Buffer:

5% Horse Serum in PBS 

+ 0.3% Triton-X

             For 25 mL:

·      2.5 mL 10 X PBS

·      1.25 mL Horse Serum

·      375 µL Triton- X (from 20%Triton-X stock)

·      20.875 ddH₂0

Antibody labeling solution:

            Dilute 1° and 2° antibodies in Blocking buffer as specified below (1:00 1° and 1:500 2° for ChAT, these will change for other antibodies)

Protocol:

1.   Add blocking buffer to slices, shake at RT for 1 hr.

2.   Aspirate Block buffer (no washing necessary)

3.   Add 400 uL/well of  1° antibody (goat α-ChAT), 1:100 dilution

4.   Shake O/N at 4°

5.   Wash min. 3x in PBS, 5 minutes shaking inbetween washes

6.   Add 400 µL/well of 2°antibody (ex. α -Goat 594), 1:500 dilution

7.   Shake at RT for 2 hr

8.   Wash min. 3x in PBS

9.   Mount on slides with DAPI

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