Feb 16, 2025

Public workspaceImmunostaining Cells

 Forked from Immunostaining
DOI
dx.doi.org/10.17504/protocols.io.4r3l29eb4v1y/v1
  • 1Caltech
Chelsie Steele
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.4r3l29eb4v1y/v1
Protocol Citation: Yujie Fan 2025. Immunostaining Cells . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29eb4v1y/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 07, 2025
Last Modified: February 16, 2025
Protocol Integer ID: 117843
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This is a protocol that describes how to use antibody staining on cells
Materials

Cell culture

25-50% confluent wells of cells growing on coverslips in 24-well plates.

Buffers, Reagents, Materials

  • 4% paraformaldehyde (diluted fromReagentPierce™ 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908 ) in 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




Fixation
Fixation
Aspirate media and gently wash each well with 1X PBS.

Wash
Aspirate PBS and place Amount500 µL of 4% paraformaldehyde onto each well.

Pipetting
Fix at TemperatureRoom temperature for Duration00:10:00 .

10m
Remove 4% paraformaldehyde and add Amount1 mL 1X PBS. Repeat this wash with PBS three times.



Pipetting
Immunostaining
Immunostaining
13h 55m
13h 55m
Permeabilize Amount1 mL PBS + 0.1% Triton X-100 (PBST) and gently shake at TemperatureRoom temperature for Duration00:10:00 .

10m
Pipetting
Prepare 5% NDS blocking buffer (for instance, to prepare 10mL of buffer, add 500uL NDS stock to 9.5mL of PBST). Block in Amount1 mL Blocking Buffer for Duration01:00:00 . Add enough blocking buffer to fully cover the well (about 500uL in a 12-well plate).

1h
Pipetting
Dilute primary antibodies into NDS Blocking Buffer and add Amount300-500 µL of diluted antibodies to each well.

Pipetting
Leave in Temperature4 °C DurationOvernight .

12h
Overnight
Wash the cells 3x with Amount1 mL PBS + 0.1% Triton X-100 at TemperatureRoom temperature each time for Duration00:10:00 .

10m
Pipetting
Wash
Dilute secondary antibodies into Blocking Buffer at a 1:500 diltion.

Pipetting
Incubate cells on coverslips in Amount300-500 µL diluted secondary antibody for 1-2 hr at TemperatureRoom temperature protected from light.

Incubation
Pipetting
Wash with Amount1 mL PBS + 0.1% Triton X-100 for Duration00:10:00

10m
Pipetting
Wash
Add Amount1-10 µL DAPI and incubate with gentle shaking for Duration00:10:00 , protected from light.

10m
Incubation
Pipetting
Wash 2x with Amount1 mL PBS + 0.1% Triton X-100 for Duration00:10:00

10m
Wash
Allow mounting medium to come to TemperatureRoom temperature .

Add one drop of mounting medium onto microscope slides for each coverslip.
Pipetting
Mount coverslips onto slides.
Let slides cure at TemperatureRoom temperature DurationOvernight , protected from light.

5m
Overnight
Microscopy
Microscopy
Collect images of fixed cells using a microscope:
  • For high resolution: use Leica DMi8 Inverted Microscope with Spinning Disk