Oct 03, 2024
Immunocytochemistry
- 1Van Andel Institute
Protocol Citation: Michael Henderson, Naman Vatsa 2024. Immunocytochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82ym8l2w/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 02, 2024
Last Modified: October 09, 2024
Protocol Integer ID: 108928
Funders Acknowledgement:
Aligning Science Across Parkinson's
Abstract
This is a protocol for immunocytochemistry for 24 and 96 well cell culture plates.
Materials
| A | B | C | D | |
| Vendor | Catalog# | Qty | Description | |
| RPI | A30075-100.0 | 1 | Bovine Serum Albumin | |
| Electron Microscopy Services | 15713 | 10 X 10ml | 20% Paraformaldehyde |
Preparation
Preparation
Prepare 1x phosphate buffer saline (PBS)
Prepare fixation solution: 4% paraformaldehyde/4% sucrose in PBS
Note
can be stores at 4°C for up to 1 month
Prewarm the fixative solution to 37 degrees Celsius
Prepare blocking buffer: 3% Bovine Serum Albumin (BSA) in PBS
- Seal the conical with parafilm to prevent evaporation and store at 4 degrees Celsius
Prepare 10% stock of Triton-X-100 in PBS
Prepare permeabilization buffer: 0.3% TX-100 in 3% BSA in PBS
Staining
Staining
Thoroughly aspirate media from each well without disturbing cells
Add pre-warmed fixative solution to the wells and incubate at room temperature for 15 minutes. (0.5 mL/well for 24-well plate, 100µL/well for 96-well plate).
Rinse 5X with PBS. This and subsequent washes can be done with the plate washer for 96-well plates.
Permeabilize neurons with a permeabilization buffer for 15 minutes at room temperature.
Rinse 3X with PBS.
Block neurons for a further 50 minutes with a blocking buffer at room temperature.
Dilute primary antibodies in blocking buffer.
Incubate cells with primary antibody for 2 hours at room temperature or overnight at 4°C.
Rinse 5X with PBS.
Dilute Alexa Fluor conjugated secondary antibodies corresponding to primary antibodies
in blocking buffer in 1:500 dilution
Incubate cells in secondary antibodies for 1 hour at room temperature.
Note
Make sure that the secondary antibodies will not recognize each other or crossreact with the same primary antibodies!
Rinse 5X with PBS.
Mount coverslips onto glass slides with Prolong Gold mounting media. 96-well should be
incubated with 100 µL DAPI 1:10,000 in PBS.
Seal the 96-well plate with a black seal.
Visualize staining on a fluorescent microscope.