Jul 13, 2024

Public workspaceImmunohistochemistry for anti-GFP

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lywe8pvx9/v1
  • Lauren Faget1,
  • Thomas Hnasko1
  • 1University of California, San Diego
Lauren C. Faget
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lywe8pvx9/v1
Protocol Citation: Lauren Faget, Thomas Hnasko 2024. Immunohistochemistry for anti-GFP. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywe8pvx9/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: July 12, 2024
Last Modified: July 13, 2024
Protocol Integer ID: 103347
Keywords: immunohistochemistry, gfp
Abstract
Hnasko lab protocol for anti-GFP immunohistochemistry
Protocol:
Day 1
Wash with 1X PBS (Phosphate Buffered Saline, 10X solution, Fisher bioreagents, BP399-1) Ph7.4 Duration00:05:00 3 times

5m
Wash with 1X PBS containing 0.2% Triton X-100 (Sigma-Aldrich, X100-1L) (PBS-T). Duration00:05:00 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. Duration01:00:00 minimum at TemperatureRoom temperature

1h
Incubate with primary antibodies in PBS-T with 4% NDS.
DurationOvernight at Temperature4 °C
Wrap well-plate air-tight with parafilm and cover with foil.
Rabbit anti-GFP polyclonal antibody, Invitrogen, A11122 (*Upon arrival: Diluted 1:1 in glycerol by adding same volume 100% glycerol, and stored at -20C). Dilution: 1:1000 to have a final dilution of 1:2000. Example: Amount1 µL anti-GFP + Amount999 µL PBS-T 4% NDS

Day 2
Wash with 1X PBS-T. Duration00:10:00 3 times

10m
Incubate with secondary antibodies in 1X PBS-T.
Duration02:00:00 at TemperatureRoom temperature
Cover well-plate with foil.
AlexaFluor 488 Donkey anti-rabbit, Jackson ImmunoResearch, 711-545-152 (*Upon arrival: Reconstituted with ~0.4 ml 50% glycerol and stored at -20C). Dilution: 1:400.
Example: Amount1 µL D@Rb488 + Amount399 µL PBS-T

2h
Wash with 1X PBS. Duration00:10:00 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 ~Amount120 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:
Use a 24-well plate. Place a maximum of 12 sections / well. Use Amount1 mL / well for washes and blocking. Use from Amount500 µL to Amount750 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:
Discard any sections found stuck to side of wells

Samples:
30um-sections in 48 well-plates at Temperature4 °C in 1X PBS with 0.01% Sodium Azide (Fisher Scientific, BP922I-500) solution

Purpose:
GFP or YFP amplification. This protocol can also be applied to other epitopes/antibodies.