Immunostaining

Cole S Sitron; Victoria A Trinkaus; F Ulrich Hartl

Jan 22, 2024

Immunostaining

DOI

dx.doi.org/10.17504/protocols.io.e6nvwd7n7lmk/v1

Cole S Sitron¹,
Victoria A Trinkaus¹,
F Ulrich Hartl¹

¹Department of Cellular Biochemistry, Max Planck Institute of Biochemistry

Cole Sitron

Max Planck Institute of Biochemistry

DOI: dx.doi.org/10.17504/protocols.io.e6nvwd7n7lmk/v1

Protocol Citation: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl 2024. Immunostaining. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd7n7lmk/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 16, 2024

Last Modified: May 31, 2024

Protocol Integer ID: 93738

Keywords: ASAPCRN

Funders Acknowledgement:

Aligning Science Across Parkinson's

Grant ID: ASAP-000282

Abstract

This is a protocol that describes how to use antibody staining to detect cellular epitopes by immunofluorescence microscopy.

Attachments

260-2491.docx

16KB

Materials

Cell culture

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

Equipment

Platform shaker
Microcentrifuge

Buffers & Reagents

4% paraformaldehyde (diluted fromReagentPierce&trade; 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908 ) in 1X PBS (diluted from ReagentPBS (10X), pH 7.4Thermo Fisher ScientificCatalog #70011051 )
1X PBS
0.1 % Triton X-100 in PBS
Blocking Buffer:
AB
Milk powder (Sucofin)5%
Triton X-1000.1%
PBSpH 7.4

2 drops/ml ReagentNucBlue&trade; Fixed Cell ReadyProbes&trade; ReagentThermo FisherCatalog #R37606  
ReagentFluorescence Mounting MediumAgilent TechnologiesCatalog #S302380-2  
Appropriate primary and fluorophore-conjugated secondary antibodies
Microscope slides

Fixation

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

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

Fix at TemperatureRoom temperature   for Duration00:10:00  .

10m

Remove 4% paraformaldehyde and add Amount1 mL   1X PBS.

Note
At this point, the plate can be stored for several months at Temperature4 °C  .

Immunostaining

2h 40m

Permeabilize Amount1 mL   0.1% Triton X-100 and gently shake at TemperatureRoom temperature   for Duration00:05:00  .

Block in Amount1 mL   Blocking Buffer for Duration01:00:00  .

Dilute primary antibodies into Blocking Buffer and add Amount300 µL   of diluted antibodies to each well.

Shake at Temperature4 °C   DurationOvernight  .

Wash 3x with Amount1 mL   PBS, gently shaking at TemperatureRoom temperature   each time for Duration00:05:00  .

During washes, centrifuge tubes of secondary antibodies at Centrifigation16000 x g  for Duration00:10:00   at Temperature4 °C  .

10m

Dilute secondary antibodies into Blocking Buffer 1:500.

Incubate coverslips in Amount300 µL   diluted secondary antibody for 2-3 hr at TemperatureRoom temperature   with gentle shaking, protected from light.

Wash with Amount1 mL   PBS for Duration00:05:00   with gentle shaking.

Add Amount1 mL   diluted NucBlue and incubate with gentle shaking for Duration00:05:00  , protected from light.

Wash 2x with Amount1 mL   PBS for Duration00:05:00   with gentle shaking.

Allow fluorescence mounting medium to come to TemperatureRoom temperature  .

Add one drop of fluorescence mounting medium onto microscope slides for each coverslip.

Mount coverslips onto slides.

Let slides cure at TemperatureRoom temperature   DurationOvernight  , protected from light.

Public workspaceImmunostaining

Immunostaining