Oct 28, 2024
Immunohistochemistry for anti-TH
Forked from Immunohistochemistry for anti-GFP
- 1University of California, San Diego;
- 2University of California San Diego
Protocol Citation: Lauren C. Faget, Thomas Hnasko, Grace Kollman 2024. Immunohistochemistry for anti-TH. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9xq9v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 24, 2024
Last Modified: October 28, 2024
Protocol Integer ID: 110864
Abstract
Hnasko lab protocol for anti-TH immunohistochemistry
Protocol:
Protocol:
Day 1
Wash with 1X PBS (Phosphate Buffered Saline, 10X solution, Fisher bioreagents, BP399-1) 7.4 00:05:00 3 times
5m
Wash with 1X PBS containing 0.2% Triton X-100 (Sigma-Aldrich, X100-1L) (PBS-T). 00:05:00 3 times
5m
Blocking step with appropriate normal serum (use serum from the source species for the secondary antibodies to prevent nonspecific binding).
Incubate with 4% NDS (Normal Donkey Serum, Jackson ImmunoResearch, 017-000-121) in PBS-T. 01:00:00 minimum at Room temperature
1h
Incubate with primary antibodies in PBS-T with 4% NDS.
Overnight at 4 °C
Wrap well-plate air-tight with parafilm and cover with foil.
Sheep anti-TH polyclonal antibody, Pel-Freez, P60101-150. Dilution: 1:1000.
Example: 1 µL anti-TH + 999 µL PBS-T 4% NDS
Day 2
Wash with 1X PBS-T. 00:10:00 3 times
10m
Incubate with secondary antibodies in 1X PBS-T.
02:00:00 at Room temperature
Cover well-plate with foil.
Alexa 647 Donkey anti-sheep, Jackson ImmunoResearch, 713-605-147 (*Upon arrival: dilute 1:1 with 100% glycerol for a final concentration of 50% glycerol and store at -20). Dilution: 1:400.
Example: 1 µL D@Sh647 + 399 µL PBS-T
2h
Wash with 1X PBS. 00:10:00 3 times
10m
Mount on Slides (22-034-979, Fisherbrand). Takes practice!
Let dry on a slide holder (VWR, 48429-105). Cover from light
Rinse with ddH2O. Briefly dip slide in a 50mL falcon tube filled with ddH2O
Place back on slide holder and let dry covered from light
Apply ~120 µL of Fluoromount-G Fluorescent mounting medium (SouthernBiotech, 0100-01) with 0.5 μg/ml DAPI (Millipore, 10236276001) on dried slides and apply coverslip (2980-225, Corning)
Protect from light and dry overnight before imaging
Material:
Material:
Use a 24-well plate. Place a maximum of 12 sections / well. Use 1 mL / well for washes and blocking. Use from 500 µL to 750 µL / well for antibody incubations. Use fire-shaped blunted Pasteur glass pipette or thin brush to transfer section from 1 well to another. Place well plate on shaker, rocker, or belly dancer for all washes and incubation steps.
Note:
Note:
Discard any sections found stuck to side of wells
Samples:
Samples:
30um-sections in 48 well-plates at 4 °C in 1X PBS with 0.01% Sodium Azide (Fisher Scientific,
BP922I-500) solution
Purpose:
Purpose:
Visualization of TH/dopmaine neurons. This protocol can also be applied to other epitopes/antibodies.