006005004134.GOT
Date: 27-MAR-2024
Author: IB,JH. Version: 3

Immunofluorescence
ProtocolValidated for all CELLINK bioinks, including the alginate based, nanofibrillated
cellulose based, collagen based, and GelMA based bioinks. This is a suggested
procedure, please adjust according to your experimental needs.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions for
immunofluorescence staining of paraffin embedded,
sectioned constructs. Follow Paraffin Embedding
Protocol and Sectioning Protocol before starting thisprotocol.

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Antigen retrieval buffer (Tri-sodium citrate,distilled water, sodium hydroxide/acetic acid,Triton 100X/Tween20)
Distilled water
Bovine serum albumin (BSA) or serum of hostof secondary antibody
PBS/TBS
Triton 100X or Tween20
Microscope slides with sectioned construct
96% Ethanol
100% Ethanol
Xylene or xylene substitute, e.g. Shandon
Xylene Substitute (ThermoFisher, Ref:9990505)
Water bath that can reach 98-100°C
Microscope slide container
Super PAP pen
Primary antibody
Secondary antibody
DAPI (1 mg/mL)
Cover glass
Mounting medium, e.g., Fluoromount G

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Protocol
All handling and use of ethanol and xylene/xylene substitute must be done inside a fume hood with proper PPE
and waste disposed according to local regulation.

1.

Preparing antigen retrieval buffer

MATERIAL
Dihydrate tri-sodium citrate
Distilled water
Sodium hydroxide/acetic acid
Triton 100X/Tween20

DESCRIPTION
Note: If antigen retrieval buffer already is prepared, proceed to Step 2.
•Prepare antigen retrieval buffer by:
1.
2.
3.
4.

•Diluting 2.94 g of dihydrate tri-sodium citrate in 1000 mL of distilled water.
Adjust the pH to 6.0 with sodium hydroxide/acetic acid.
Add 0.5 mL of Tween 20/Triton 100x.
Mix the solution properly.

Antigen retrieval buffer can be stored 1 month in room temperature, for extended storage store at 4°C.

2.

Preparing blocking solution

MATERIAL
BSA or serum of host of secondary antibody
PBS/TBS
Triton 100X/Tween20

DESCRIPTION
Note: If blocking solution already is prepared, proceed to Step 3.
•3.

Prepare a 3% blocking solution by dissolving 1.5 g of BSA or serum of host of secondary antibody in 50mL PBS or TBS. Add 50 µL Triton 100X /Tween20 for a 0.1% final concentration. Store at 4°C.

Deparaffination and re-hydration

MATERIAL
Microscope slides with sectioned construct
Xylene or xylene substitute
100% ethanol
Distilled water
96% ethanol

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DESCRIPTION•

Deparaffinize and rehydrate sections by moving microscope slides with sectioned construct throughfollowing series of liquids:
1.
2.
3.
4.

4.

Xylene or xylene substitute: 3 x 5 min
100% ethanol: 1 min
96% ethanol: 1 min
Distilled water: at least 2 min

Antigen retrieval

MATERIAL
Antigen retrieval buffer
Water bath
Microscope slide container (stays intact at 100°C)

DESCRIPTION••••

Put the microscope slides in the container and fill up with antigen retrieval buffer until the slides arecovered.
Place the container with samples and retrieval buffer in the cold water bath.
Turn on heating and let the water reach boiling (98°C-100°C). Let the samples incubate 10 min, startingfrom when the water starts boiling (above 98°C ok).
Let the samples cool in the buffer.

Note: To speed up cooling, put the container with samples and retrieval buffer in cold water after treatment.

5.

Preparation of samples

MATERIALPBS/TBS
Super PAP pen

DESCRIPTION••

6.

Rinse slides 2 x 3 min in PBS/TBS
Let slides dry and circle the sections with the Super PAP pen to separate/highlight them. This allows for
multiple staining on the same slide.

Blocking/permeabilization

MATERIAL
Blocking solution

DESCRIPTION••

Add blocking solution to the sections, incubate 45 min at room temperature.
Blot of blocking solution, do not rinse.

Note: Always add enough solution to completely cover the sections.

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7.

Primary antibody

MATERIAL
Microscope slide box
Primary antibody
Blocking solution
PBS/TBSDESCRIPTION
••
•
•••

Make a humified chamber of the microscope box by adding wet paper at the bottom of the box.
Place slides horizontally in the humified chamber.
Dilute primary antibody in blocking solution at recommended concentration, calculate 40-50 µL persection.
Add enough primary antibody to completely cover the sections.
Place horizontally in humified chamber over night at 4°C or in room temperature for 60 min.
Rinse 2 x 3 min with PBS/TBS.

8.

Secondary antibody

MATERIAL
Microscope slide box
Secondary antibody
Blocking solution

DESCRIPTION•••••

Make sure to work in darkness to not bleach the fluorophores.
Blot off excess PBS/TBS, it’s important to not dilute secondary antibody.
Dilute the secondary antibody in blocking solution at recommended concertation, calculate 40-50 µL persection.
Add enough secondary antibody to completely cover the sections.
Incubate 60 min in humified chamber at room temperature.

9.

DAPI stainMATERIALDAPI (1 mg/mL)
PBS/TBSDESCRIPTION•••••

Make sure to work in darkness to not bleach the fluorophores.
Dilute DAPI (1 mg/mL) 1:50 in PBS/TBS.
Blot off secondary antibody and add enough DAPI to completely cover the sections.
Incubate 10 min in room temperature in humified chamber.
Rinse 2 x 3 min with PBS/TBS.

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10.

Prepare slides

MATERIAL
Mounting medium
Cover glass

DESCRIPTION•••

Blot of the PBS/TBS and apply a drop of mounting medium to the stained slides.
Cover with a cover glass, apply carefully to avoid air bubbles.
Let dry at 4°C horizontally overnight.

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