Feb 17, 2025
Immunostaining Mouse Tissue
- 1Caltech
Protocol Citation: Chelsie Steele, Yujie Fan 2025. Immunostaining Mouse Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q7ypv25/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: January 08, 2025
Last Modified: February 17, 2025
Protocol Integer ID: 117844
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol describes immunohistochemical and immunoctyochemical methods to analyze protein expression in mouse brain. In the protocol, we describe the fixation of tissue or cultured neurons, the immunostaining procedure, and collecting images for analysis.
Materials
Buffers, Reagents, Materials
- 4% paraformaldehyde (diluted fromPierce™ 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908 ) in 1X DPBS
- 1X DPBS
- 0.1 % Triton X-100 in DPBS
- Blocking Buffer: Normal Donkey Serum -Jackson Immune Research Labs - Cat No NC9624464
- DAPI - ThermoFisher Cat No D1306
- Prolonged Diamond Antifade Mountant - ThermoFisher Cat No P36965
- Appropriate primary and fluorophore-conjugated secondary antibodies
- Microscope slides
Collection of fixed tissues slices from mice
Collection of fixed tissues slices from mice
1d 0h 10m
1d 0h 10m
Anesthetize mouse, pin onto dissection tray and open chest cavity
Using a perfusion pump, pierce needle into left ventricle and sever the right atrium; immediately begin perfusing cold 1X PBS followed by 4% paraformaldehyde (PFA) in PBS
Dissect out brain or wanted tissue into 15 mL tube containing cold 4% PFA in PBS
Incubate Overnight at 4 °C in 4% PFA in PBS
12h
Incubate Overnight at 4 °C in 30% sucrose in PBS
12h
Tissue can be kept in long-term storage at -80 °C ; when ready for IHC, slice 100 μm sections using a cryostat (Leica CM3050 S)
Equipment
Cryostat
NAME
Leica
BRAND
Cryostat
SKU
LINK
Once slices are collected, IHC is performed on free-floating sections in cold 1X DPBS with 5% Azide
Tissue staining
Tissue staining
18h
18h
Wash samples three times for 10 minutes at Room temperature in 1X DPBS + 0.1% Triton X-100 on a rocker
30m
Block and permeabilize for 2 hours at Room temperature in 1X DPBS containing 4% normal goat serum (NGS) and 0.1% Triton X-100 on a rocker
2h
Prepare primary antibody solution by diluting antibody at desired concentration in 1X DPBS containing 2% NGS and 0.1% Triton X-100
Incubate in primary antibody overnight at 4 °C
12h
Wash samples three times for 10 minutes at Room temperature in 1X DPBS+ 0.1% Triton X-100 on a rocker
30m
Prepare secondary antibody solution by diluting antibody at 1:1000 in 1X DPBS containing 2% NGS and 0.1% Triton X-100
Incubate in secondary antibody for 2 hours at 4 °C in the dark
2h
Wash samples three times for 10 minutes at Room temperature in 1X PBS + 0.1% Triton X-100 on a rocker
30m
Add DAPI 1:1000 to free floating samples for 10 minutes
10m
Wash samples two times for 10 minutes at RT in 1X DPBS + 0.1% Triton X-100 on a rocker
20m
Mount free floating slices onto a slide.
- For all sample types, mount using Prolonged Diamond Antifade Mountant
Store samples at 4 °C for short-term storage or -20 °C for long-term storage
Imaging
Imaging
Collect images of fixed tissue using a microscope:
- For high resolution: use Leica DMi8 Inverted Microscope with Spinning Disk