Jan 30, 2019
Basic immunofluorescence protocol for adherent cells
- 1Wyss Institute for Biologically Inspired Engineering, Harvard University
Protocol Citation: Girija Goyal 2019. Basic immunofluorescence protocol for adherent cells. protocols.io https://dx.doi.org/10.17504/protocols.io.wt4feqw
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, 2019
Last Modified: January 30, 2019
Protocol Integer ID: 19036
Abstract
Adherent cells were cultured in flat bottom plates and fixed in situ. This is a protocol to detect specific proteins by immunofluorescence in these cells. Immunofluoroscence is a common laboratory technique where specific proteins within cells can be detected using antibodies coupled to fluorophores and then visualized using a microscope. The cells were counterstained with Hoechst dye to label nuclei.
Guidelines
Secondary antibodies usually target species specific epitopes on the primary antibody. Thus, if the primary antibody was raised in goats, then the secondary should say "anti-goat". Further, antibodies can be of different isotypes such as IgM or IgG kappa (k) vs. lambda, or IgG1 vs. IgG2. It is important to make sure that the chosen secondary detects the specific isotype of your primary or all isotypes (for example, anti-goat IgG will detect both IgG1 and IgG2 or anti-goat Ig will all goat antibodies).
Anti-luciferase antibody
for 96 well plate, use 100 µL of buffers
Materials
MATERIALS
Triton X-100 Bio-Rad LaboratoriesCatalog #1610407
PBS
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterInvitrogen - Thermo FisherCatalog #H3570
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
STEP MATERIALS
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
Protocol materials
Triton X-100 Bio-Rad LaboratoriesCatalog #1610407
PBS
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterInvitrogen - Thermo FisherCatalog #H3570
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
Before start
1. Make sure the stock of paraformaldehyde (PFA) has not expired and is dissoved in an isotonic buffer such PBS.
2. Prepare or check that you have enough of the following buffers
a. Permeabilization buffer: 0.1% Titron X, 1-2%FBS or BSA in PBS
b. Staining buffer without detergent: 1-2%FBS or BSA in PBS
Fixation
Fixation
Fix the cells using 4% paraformaldehyde (PFA) by removing media and submerging cells in PFA for 00:15:00 at 20 °C room temp . Cells can be left in fixative for a few days at 4 °C Fridge
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
Aspirate fixative and replace with PBS. Cells can be stored at this step for upto 1 month.
Permeabilization
Permeabilization
For the antibody to be able to penetrate the cells, the membranes need to be permeabilized. This may be necessary even if the protein to be detected is expressed on the surface if the antibody binds an intracellular region. Aspirate PBS from previous step and replace with 100 µL permeabilization buffer and incubate for00:10:00 20 °C Room temp
Store staining buffer in 4 °C fridge
Aspirate permeabilization buffer and replace with 100 µL PBS. Aspirate PBS and proceed to next step or store in the fridge in fresh PBS.
Primary antibody staining
Primary antibody staining
Incubate in primary antibody (the antibody that detects the protein of interest) at the recommended dilution (1:100) in staining buffer 00:00:00 Overnight 4 °C . See guidelines for dilutions of specific antibodies and duration of incubation.
Store staining buffer in 4 °C fridge
If you plan to reuse the diluted antibody for another experiment, use a pipette to transfer it into a collection tube for storage. Otherwise, aspirate primary antibody from the cells, and add PBS and leave plate on shaker for 00:05:00 at room temp. Repeat twice.
Secondary staining
Secondary staining
For greater flexibility, primary antibodies are left unconjugated and detected using a secondary antibody which is specific to primary antibody (see guidelines) and are labeled with a fluorophore. Incubate in secondary antibody at the recommended dilution in staining buffer. See guidelines for dilutions of specific antibodies and duration of incubation.
Post-secondary washing and counterstaining for nuclei/DNA
Post-secondary washing and counterstaining for nuclei/DNA
Aspirate secondary antibody from the cells, and add PBS and leave plate on shaker for 00:05:00 at room temp.
Replace PBS with 1:2000 dilution of Hoechst dye and incubate at room temp for 00:20:00
Conduct two washes with PBS as in step 8. The cells are ready for visualization on the microscope.