May 05, 2025

Public workspaceFIMM Digital Microscopy and Molecular Pathology Core Unit

DOI
dx.doi.org/10.17504/protocols.io.rm7vz6775gx1/v1
  • 1University of Helsinki
Teijo Pellinen
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DOI: dx.doi.org/10.17504/protocols.io.rm7vz6775gx1/v1
Protocol Citation: Teijo Pellinen, Nora Linnavirta, annabrita.schoonenberg, Katja Välimäki 2025. FIMM Digital Microscopy and Molecular Pathology Core Unit. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz6775gx1/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: April 01, 2025
Last Modified: May 05, 2025
Protocol Integer ID: 131026
Keywords: FIMM Digital Microscopy, Molecular Pathology Core Unit, AlexaFluor stainings, Antigen retrieval, Antibodies detection
Funders Acknowledgements:
Research Council of Finland
Grant ID: 363152
Abstract
This protocol describes a multiplex immunofluorescence workflow developed at the FIMM Digital Microscopy and Molecular Pathology Core Unit for profiling formalin-fixed, paraffin-embedded tissue sections. It combines dual-color tyramide signal amplification (TSA) with iterative rounds of antibody stripping and non-TSA HRP-based secondary detection to visualize six or more protein markers on a single slide. Key steps include slide mounting and drying; deparaffinization and rehydration; heat-induced antigen retrieval; endogenous peroxidase blocking; two sequential TSA reactions; heat-mediated antibody denaturation; and up to four additional HRP-secondary staining cycles without TSA. After each cycle, slides are scanned at high resolution for automated image analysis. This highly flexible system permits user-defined marker panels, fluorophores and cycle numbers. Although staining can be extended to include more rounds, repeated stripping may eventually weaken or detach tissue, depending on sample quality. Optimized for reproducibility and seamless integration with scalable bioinformatics pipelines, this protocol provides a versatile tool for detailed spatial mapping of immune and stromal populations in the tumor microenvironment.
Materials
Protocol design
Antibodies (Ab1, Ab2…) preferably in mouse-rabbit pairs
AB
DetectionMultiplex Panel  
TSA488 Ab1 
TSA555 Ab2 
Boil 
AF647Ab3 
AF750Ab4 
scan 1Bleach and boil 
AF647Ab5  
AF750Ab6 
scan 2Bleach and boil 
AF647Ab7 
AF750Ab8 
scan3Bleach and boil 
AF647Ab9 
AF750Ab10 
scan4  
Reagents and materials
ABCD
CategoryNameCompany/ cat.Number Dilution/ lot.nr. 
rehydration Alcohol AbsoluteVWR/ 20821.365  
rehydrationAlcohol 96%VWR/ 20824.365  
rehydrationXyleneVWR/534056  
HIER100mM Tris-10mM EDTA, pH9Home madeDilute 10x in Milli-Q 
wash10x TBSHome madeDilute 10x in Milli-Q 
Tris BaseFisher BP152-5  
NaCl FisherBP358-212  
washTweenFisher/10113103  
blockH2O2Fisher/ 10736291  
blockNormal goat serum (NGS)Gibco/ 16210-064  
secondary antibodiesG-a-M-AF 647Life/ A21236  
secondary antibodiesG-a-R-AF 647Life/ A21245  
secondary antibodiesG-a-M-AF 750Life/ A21037  
secondary antibodiesG-a-R-AF 750Life/ A21039  
secondary antibodiesG-a-M-poly HRPImmunologic/ DPVM55HRP  
secondary antibodiesG-a-R-poly HRPImmunologic/ DPVR110HRP  
Fluorescent substrateTyramide 488Life/ B40953   
Fluorescent substrateTyramide 555Life/ B40955  
Counter stainingDapiRoche/ 10236276001 
Mounting medium (fluorescence)Prolong GoldLife/ P36934  
Microscope slidesSuperFrost Plus/ Fisher J3800AMNZ or similar  
PT ModuleThermo Scientific  
Fluorescent scanner3D Histech; Pannoramic Zeiss; Axioscan z.1
Buffer/ reagent Preparation

Xylene/ alcohol series
Change the solutions every month!

Wash buffers
TBS: Prepare 10x TBS Ph7.6 from Tris-Base and NaCl. Store at TemperatureRoom temperature max 6 months. Dilute 10x with Milli-Q to 1x TBS buffer, store at TemperatureRoom temperature max 1 week.
TBST: Dilute 10x TBS in Milli-Q and add Amount1 mL 50% Tween (in Milli-Q)/ Amount1 L (end conc. tween= 0.05%), store at TemperatureRoom temperature max 1 week.

Endogenous peroxidase block
0.9% H2O2 in TBS
AB
TBS250 ml
30% H2O27.5 ml

Protein block: 10% NGS in TBST
AB
TBST45 ml
NGS (fresh aliquot from -20°C)5 ml
Mix and filter (low protein binding Thikness0.22 µm or Thikness0.45 µm ), store at Temperature4 °C , max 1 week

Primary Antibodies:
Dilute in 10% NGS in TBST

Secondary Antibodies:
Dilute in TBST

TSA (tyramide) reagents:
Prepare 0.3% H2O2 just before use!
Dilute 30% H2O2 stock 1:100 in TBST!
For Amount100 µL TSA reagent: add Amount0.5 µL 1:100 diluted H2O2 (final dilution 1:20.000) and Amount1 µL Tyramide fluorochrome (final dilution 1:100), mix well.
Note
Use within Duration01:00:00 , avoid exposure to light.


Bleaching solution
for 200 ml: Amount30 mL 30% H2O2+ Amount4.8 mL Concentration1 Molarity (M) NaOH, add TBS to Amount200 mL .

ReagentEthanol absolute ≥99.8%VWR International (Avantor)Catalog #20821.365
ReagentEthanol GPR RECTAPUR®VWR International (Avantor)Catalog #20824.365
ReagentXylene (mixture of isomers) for histologyVWR International (Avantor)Catalog #534056-4L
ReagentTris Base (White Crystals or Crystalline Powder/Molecular Biology)Fisher ScientificCatalog #BP152-5
ReagentSodium ChlorideFisher ScientificCatalog #BP358-212
ReagentPolysorbate 20/TweenFisher ScientificCatalog #10113103
ReagentHydrogen Peroxide 30-32% (w/w) (100 Volumes), Certified AR for Analysis, Fisher Chemical™Fisher ScientificCatalog #10736291
ReagentGoat SerumGibco - Thermo Fisher ScientificCatalog #16210-064
ReagentGoat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-21236
ReagentGoat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-21245
ReagentGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 750Thermo Fisher ScientificCatalog #A-21037 ReagentGoat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 750Thermo Fisher ScientificCatalog #A-21039
ReagentBrightVision, 1 step detection system Goat Anti- Mouse HRP / Goat Anti- Rabbit APImmunologic a WellMed CompanyCatalog #DPVM55HRP ReagentBrightVision, one component detection system Goat Anti- Rabbit HRP / Mouse APImmunologic a WellMed CompanyCatalog #DPVO110-rHRP-mAP ReagentTyramide ConjugatesThermo Fisher ScientificCatalog #B40953 ReagentAlexa Fluor™ 555 Tyramide ReagentThermo FisherCatalog #B40955 ReagentDAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001 ReagentProLong™ Gold Antifade MountantThermo Fisher ScientificCatalog #P36934







Protocol
Protocol
Mount sections on microscope slides and attach by drying DurationOvernight at TemperatureRoom temperature or Temperature37 °C .
Overnight
Optional: keep slides 15-30 min at Temperature60 °C before starting paraffin removal.

Optional
Paraffin removal and rehydration
Paraffin removal and rehydration
23m
23m
Place the slides in a plastic slide holder!
Immerse the slides in xylene and alcohol solutions in the following order.
Xylene for Duration00:05:00 . (1/3)

5m
Xylene for Duration00:05:00 . (2/3)
5m
Xylene for Duration00:05:00 . (3/3)
5m
Alcohol absolute for Duration00:01:00 . (1/3)

1m
Alcohol absolute for Duration00:01:00 . (2/3)
1m
Alcohol absolute for Duration00:01:00 . (3/3)
1m
96% alcohol for Duration00:01:00 . (1/2)

1m
96% alcohol for Duration00:01:00 . (2/2)
1m
70% alcohol for Duration00:01:00 .

1m
Milli-Q water for Duration00:01:00 . (1/2)

1m
Milli-Q water for Duration00:01:00 . (2/2)
1m
Antigen retrieval
Antigen retrieval
30m
30m
HIER:
Place the slides in Concentration10 millimolar (mM) Tris and Concentration1 millimolar (mM) EDTA buffer Ph9 in the PT module.
Heat for Duration00:20:00 at Temperature99 °C , total program lasts Duration01:10:00 .

20m
Temperature
Wash Duration00:05:00 in Milli-Q and Duration00:05:00 in TBS.
Note
NOTE: The staining can be performed using an autostainer, for which the following steps of the manual protocol must be programmed to the autostainer that is used.


10m
Wash
Endogenous peroxidase block
Endogenous peroxidase block
15m
15m
Immerse the slides in 0.9% H2O2 in TBS for Duration00:15:00 at TemperatureRoom temperature .

15m
Wash.
Wash
Wash 2x 3-5 minutes in TBS.
Wash 1x TBST.
Staining protocol: First 2 antibodies, detected with TSA reagents
Staining protocol: First 2 antibodies, detected with TSA reagents
3h 45m
3h 45m
Dry the slides around the specimen using lint-free tissue.
Apply Amount100-300 µL 10% NGS in TBST per slide, make sure the specimen is covered completely.

Incubate for Duration00:15:00 at TemperatureRoom temperature .

15m
Incubation
Dilute the primary antibody or antibodies in 10% NGS in TBST.
Tap off the solution and apply Amount100-300 µL diluted primary antibody.

Incubate for Duration01:00:00 at TemperatureRoom temperature or DurationOvernight at Temperature4 °C (depending on the antibody).
1h
Incubation
Wash 3x 3-5 min TBST.
Wash
Apply Amount100-300 µL 1:5 diluted (in TBST) Bright Vision goat anti mouse or rabbit HRP per slide.

Incubate Duration00:30:00 TemperatureRoom temperature .

30m
Incubation
Wash 3x 3-5 min TBST.
Wash
Prepare the TSA-AF488 reagent just before use.
Pipet Amount100-300 µL reagent per slide and incubate Duration00:15:00 TemperatureRoom temperature .

15m
Incubation
Pipetting
Wash 3x 3-5 min TBST.
Wash
Note: In case the second primary antibody is made in the same species as the first:
  • Denature the previous antibody by heating the slides in freshly prepared Concentration10 millimolar (mM) Tris and Concentration1 millimolar (mM) EDTA Ph9 in the PT module at Temperature99 °C for Duration00:20:00 .
  • Cool in MilliQ-water 5min. Perform endogenous peroxidase block and then follow steps 8-14 of the staining protocol.
  • Continue for TSA-AF555 detection with step 24.
20m
Immerse the slides in 0.9% H2O2 in TBS for Duration00:15:00 TemperatureRoom temperature .

15m
Wash 1x 3-5 minutes in TBS and 1x TBST.
Wash
Apply Amount100-300 µL 1:5 diluted (in TBST) Bright Vision goat anti mouse or rabbit HRP per slide.

Incubate Duration00:30:00 TemperatureRoom temperature .

30m
Incubation
Wash 3x 3-5 min TBST.
Wash
Prepare the TSA-AF555 reagent just before use.
Pipet Amount100-300 µL reagent per slide and incubate Duration00:15:00 TemperatureRoom temperature .

15m
Incubation
Wash 3x 3-5 min TBST and store at Temperature4 °C DurationOvernight if not proceeding immediately.
Wash
Overnight
  • After the second TSA reaction denature the previous antibody or antibodies by heating the slides in freshly prepared Concentration10 millimolar (mM) Tris and Concentration1 millimolar (mM) EDTA Ph9 in the PT module at Temperature99 °C for Duration00:20:00 .
  • Cool in MilliQ water Duration00:05:00 .

25m
Continue to AlexaFluor staining.
Staining protocol: AlexaFluor staining
Staining protocol: AlexaFluor staining
45m
45m
Dry the slides around the tissue.
Apply Amount100-300 µL 10% NGS in TBST per slide, make sure the tissue is covered completely.

Incubate Duration00:15:00 TemperatureRoom temperature .

15m
Incubation
Dilute the primary antibodies in 10% NGS in TBST.
Tap off the blocking solution and apply Amount100-300 µL diluted primary antibodies.

Incubate 1-2h TemperatureRoom temperature or DurationOvernight Temperature4 °C .
Incubation
Overnight
Wash 3x 3-5 min TBST.
Wash
Dilute the secondary, AlexaFluor conjugated, antibodies in TBST, 1:300, add Dapi, final concentration Amount1.7 µL .

Apply Amount100-300 µL of the secondary reagent solution per slide and incubate Duration00:30:00 at TemperatureRoom temperature .
30m
Incubation
Wash 3x 3-5min TBST and 1x Duration00:05:00 Milli-Q.

Wash
Dry slides at TemperatureRoom temperature .
Mount sections in ProlongGold anti-fade mounting medium, use coverslips with thickness: no. 1.5.
Dry the slides before scanning, avoid exposure to light.
Scan/image the fluorescence using the appropriate filters.
After scanning, soak off the coverslips in TBST at TemperatureRoom temperature or Temperature4 °C .

Staining protocol: Second to fourth round AlexaFluor stainings
Staining protocol: Second to fourth round AlexaFluor stainings
45m
45m
Wash the slides after the coverslips are soaked off in TBS.
Wash
Bleach the attached AF647 and AF750 by soaking the slides in TBS/NaOH/H2O2, 30-60 min TemperatureRoom temperature .
Rinse in 1x TBS and MilliQ.
Wash
  • Denature the previous antibodies by heating the slides in freshly prepared Concentration10 millimolar (mM) Tris and Concentration1 millimolar (mM) EDTA Ph9 in the PT module at Temperature99 °C for Duration00:20:00 .
  • Cool in MilliQ-water Duration00:05:00 and wash in TBS Duration00:05:00 .

30m
Wash
Apply Amount100-300 µL 10% NGS in TBST per slide, make sure the tissue is covered completely.

Incubate Duration00:15:00 TemperatureRoom temperature .

15m
Incubation
Dilute the primary antibodies in 10% NGS in TBST.
Tap off the blocking solution and apply Amount100-300 µL diluted primary antibodies.
Incubate 1-2h TemperatureRoom temperature or DurationOvernight Temperature4 °C .

Incubation
Overnight
Wash 3x 3-5min TBST.
Wash
Dilute the secondary, AlexaFluor conjugated, antibodies in TBST, 1:300, add Dapi, final concentration Amount3.4 µL (1:150).
Apply Amount100-300 µL of the secondary reagent solution per slide and incubate 30-45 minutes at TemperatureRoom temperature .
Incubation
Wash 3x 3-5min TBST and 1x Duration00:05:00 Milli-Q.

Wash
Dry slides at TemperatureRoom temperature .

Mount sections in ProlongGold anti-fade mounting medium, use coverslips with thickness: no. 1.5.
Dry the slides before scanning, avoid exposure to light.
Scan/image the fluorescence using the appropriate filters.
Imaging
After scanning, soak off the coverslips in TBST if more rounds are stained.