Jan 22, 2024

Immunostaining

DOI
https://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
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DOI: https://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, cellular epitopes by immunofluorescence microscopy, immunofluorescence microscopy, immunofluorescence, immunostaining, cellular epitope, antibody
Funders Acknowledgements:
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
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260-2491.docx
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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 fromPierce™ 16% Formaldehyde (w/v), Methanol-freeThermo FisherCatalog #28908 ) in 1X PBS (diluted from PBS (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 NucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606
  • Fluorescence 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 500 µL of 4% paraformaldehyde onto each well.

Fix at Room temperature for 00:10:00 .

10m
Remove 4% paraformaldehyde and add 1 mL 1X PBS.

Note
At this point, the plate can be stored for several months at 4 °C .


Immunostaining
2h 40m
Permeabilize 1 mL 0.1% Triton X-100 and gently shake at Room temperature for 00:05:00 .

5m
Block in 1 mL Blocking Buffer for 01:00:00 .

1h
Dilute primary antibodies into Blocking Buffer and add 300 µL of diluted antibodies to each well.

Shake at 4 °C Overnight .

1h
Wash 3x with 1 mL PBS, gently shaking at Room temperature each time for 00:05:00 .

5m
During washes, centrifuge tubes of secondary antibodies at 16000 x g for 00:10:00 at 4 °C .

10m
Dilute secondary antibodies into Blocking Buffer 1:500.

Incubate coverslips in 300 µL diluted secondary antibody for 2-3 hr at Room temperature with gentle shaking, protected from light.

Wash with 1 mL PBS for 00:05:00 with gentle shaking.

5m
Add 1 mL diluted NucBlue and incubate with gentle shaking for 00:05:00 , protected from light.

5m
Wash 2x with 1 mL PBS for 00:05:00 with gentle shaking.

5m
Allow fluorescence mounting medium to come to Room temperature .

Add one drop of fluorescence mounting medium onto microscope slides for each coverslip.
Mount coverslips onto slides.
Let slides cure at Room temperature Overnight , protected from light.

5m