Oct 06, 2023

Public workspaceYale Murine TMC - Immunofluorescence Protocol

DOI
dx.doi.org/10.17504/protocols.io.8epv5xenng1b/v1
  • 1Dept. of Pathology, Yale School of Medicine
Hester Doyle
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5xenng1b/v1
Protocol Citation: Yun-Hee Youm, Yufeng Liu, Vishwa Deep Dixit 2023. Yale Murine TMC - Immunofluorescence Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xenng1b/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 06, 2023
Last Modified: October 06, 2023
Protocol Integer ID: 88929
Abstract
Immunofluorescence staining of mouse tissues
Immunofluorescence Staining
Immunofluorescence Staining
Take out the slides to a slide holder and warm slides on a 37°C heater for 5-10 min. Grouping: usually maximum two antibodies (with secondary antibody red + green) + nuclear staining for one slide. Set negative control for each secondary antibody to detect any non-specific background.
Label slides with pencil and circle section with Pap Pen (hydrophobic marker). Move the slides into a humidified chamber. Slides should remain in humidified chamber for all the following incubation steps.
Fixation: Add one or two drops of 4% parformaldehyde in PBS on the tissue for 15-30 min.
Wash with 0.5% Tween 20 in PBS (PBST) , 10 min x 2.
Block with Blocking buffer: 10% host serum + 2% FBS in 0.5% PBST. Incubate 1 hour at room temperature or 4°C overnight.
Incubate with primary antibody: 1-10 μg/mL of purified antibody or 1:100 to 1:1000 dilution of anti-serum in 2% host serum + 2% FBS 0.5% PBST buffer overnight at 4°C. Normally start with a dilution at 1:100. For color-conjugated FC antibodies used in immunofluorescence, start at 1:50.
Wash with 0.5% PBST, 10 min x 2.
Incubate with secondary antibody: 1-10 μg/mL of purified antibody or 1:100-1:1000 of anti-serum in Blocking buffer (normally start at 1:200). Incubate 1 h at room temperature (AVOID LIGHT).
Wash with PBST, 10 min x 3.
Incubate the slides in 0.1 μg/mL DAPI for 5 min.
Wash with PBST twice before mounting.
Clean the slides.
Mount the slides with coverslips using anti-fade mounting medium.
Seal the edges of the coverslip with nail polish and let it dry.