May 05, 2025
FIMM Digital Microscopy and Molecular Pathology Core Unit
- Teijo Pellinen1,
- Nora Linnavirta1,
- Annabrita Schoonenberg1,
- Katja Välimäki1
- 1University of Helsinki
External link: https://doi.org/10.7150/thno.118400
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
Manuscript citation:
Pellinen T, Luomala L, Mattila KE, Hemmes A, Välimäki K, Arjama M, Brück O, Paavolainen L, Kankkunen E, Nisén H, Järvinen P, Castillon L, Vanharanta S, Vainio P, Kallioniemi O, Jaakkola PM, Mirtti T (2026) Tumor cell FAP orchestrates EMT and immune suppression in aggressive localized ccRCC. Theranostics 16(7). doi: 10.7150/thno.118400
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, multiplex immunofluorescence workflow, molecular pathology core unit this protocol, fimm digital microscopy, more protein marker, molecular pathology core unit, iterative rounds of antibody, antibody denaturation, mediated antibody denaturation, antibody, secondary staining cycles without tsa, antigen retrieval, seamless integration with scalable bioinformatics pipeline, scalable bioinformatics pipeline, secondary staining cycle, profiling formalin, induced antigen retrieval, tumor microenvironment, embedded tissue section, marker, immunofluorescence
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
| A | B | |
| Detection | Multiplex Panel | |
| TSA488 | Ab1 | |
| TSA555 | Ab2 | |
| Boil | ||
| AF647 | Ab3 | |
| AF750 | Ab4 | |
| scan 1 | Bleach and boil | |
| AF647 | Ab5 | |
| AF750 | Ab6 | |
| scan 2 | Bleach and boil | |
| AF647 | Ab7 | |
| AF750 | Ab8 | |
| scan3 | Bleach and boil | |
| AF647 | Ab9 | |
| AF750 | Ab10 | |
| scan4 |
Reagents and materials
| A | B | C | D | |
| Category | Name | Company/ cat. | Number Dilution/ lot.nr. | |
| rehydration Alcohol Absolute | VWR/ 20821.365 | |||
| rehydration | Alcohol 96% | VWR/ 20824.365 | ||
| rehydration | Xylene | VWR/534056 | ||
| HIER | 100mM Tris-10mM EDTA, pH9 | Home made | Dilute 10x in Milli-Q | |
| wash | 10x TBS | Home made | Dilute 10x in Milli-Q | |
| Tris Base | Fisher BP152-5 | |||
| NaCl Fisher | BP358-212 | |||
| wash | Tween | Fisher/10113103 | ||
| block | H2O2 | Fisher/ 10736291 | ||
| block | Normal goat serum (NGS) | Gibco/ 16210-064 | ||
| secondary antibodies | G-a-M-AF 647 | Life/ A21236 | ||
| secondary antibodies | G-a-R-AF 647 | Life/ A21245 | ||
| secondary antibodies | G-a-M-AF 750 | Life/ A21037 | ||
| secondary antibodies | G-a-R-AF 750 | Life/ A21039 | ||
| secondary antibodies | G-a-M-poly HRP | Immunologic/ DPVM55HRP | ||
| secondary antibodies | G-a-R-poly HRP | Immunologic/ DPVR110HRP | ||
| Fluorescent substrate | Tyramide 488 | Life/ B40953 | ||
| Fluorescent substrate | Tyramide 555 | Life/ B40955 | ||
| Counter staining | Dapi | Roche/ 10236276001 | ||
| Mounting medium (fluorescence) | Prolong Gold | Life/ P36934 | ||
| Microscope slides | SuperFrost Plus/ Fisher J3800AMNZ or similar | |||
| PT Module | Thermo Scientific | |||
| Fluorescent scanner | 3D Histech; Pannoramic Zeiss; Axioscan z.1 |
Buffer/ reagent Preparation
Xylene/ alcohol series
Change the solutions every month!
Wash buffers
TBS: Prepare 10x TBS 7.6 from Tris-Base and NaCl. Store at Room temperature max 6 months. Dilute 10x with Milli-Q to 1x TBS buffer, store at Room temperature max 1 week.
TBST: Dilute 10x TBS in Milli-Q and add 1 mL 50% Tween (in Milli-Q)/ 1 L (end conc. tween= 0.05%), store at Room temperature max 1 week.
Endogenous peroxidase block
0.9% H2O2 in TBS
| A | B | |
| TBS | 250 ml | |
| 30% H2O2 | 7.5 ml |
Protein block: 10% NGS in TBST
| A | B | |
| TBST | 45 ml | |
| NGS (fresh aliquot from -20°C) | 5 ml |
Mix and filter (low protein binding 0.22 µm or 0.45 µm ), store at 4 °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 100 µL TSA reagent: add 0.5 µL 1:100 diluted H2O2 (final dilution 1:20.000) and 1 µL Tyramide fluorochrome (final dilution 1:100), mix well.
Note
Use within 01:00:00 , avoid exposure to light.
Bleaching solution
for 200 ml: 30 mL 30% H2O2+ 4.8 mL 1 Mass Percent NaOH, add TBS to 200 mL .
Ethanol absolute ≥99.8%VWR International (Avantor)Catalog #20821.365
Ethanol GPR RECTAPUR®VWR International (Avantor)Catalog #20824.365
Xylene (mixture of isomers) for histologyVWR International (Avantor)Catalog #534056-4L
Tris Base (White Crystals or Crystalline Powder/Molecular Biology)Fisher ScientificCatalog #BP152-5
Sodium ChlorideFisher ScientificCatalog #BP358-212
Polysorbate 20/TweenFisher ScientificCatalog #10113103
Hydrogen Peroxide 30-32% (w/w) (100 Volumes), Certified AR for Analysis, Fisher Chemical™Fisher ScientificCatalog #10736291
Goat SerumGibco - Thermo Fisher ScientificCatalog #16210-064
Goat anti-Mouse IgG (H L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-21236
Goat anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-21245
Goat anti-Mouse IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 750Thermo Fisher ScientificCatalog #A-21037
Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 750Thermo Fisher ScientificCatalog #A-21039
BrightVision, 1 step detection system Goat Anti- Mouse HRP / Goat Anti- Rabbit APImmunologic a WellMed CompanyCatalog #DPVM55HRP
BrightVision, one component detection system Goat Anti- Rabbit HRP / Mouse APImmunologic a WellMed CompanyCatalog #DPVO110-rHRP-mAP
Tyramide ConjugatesThermo Fisher ScientificCatalog #B40953
Alexa Fluor™ 555 Tyramide ReagentThermo FisherCatalog #B40955
DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001
ProLong™ Gold Antifade MountantThermo Fisher ScientificCatalog #P36934
Protocol
Mount sections on microscope slides and attach by drying Overnight at Room temperature or 37 °C .
Optional: keep slides 15-30 min at 60 °C before starting paraffin removal.
Paraffin removal and rehydration
23m
Place the slides in a plastic slide holder!
Immerse the slides in xylene and alcohol solutions in the following order.
Xylene for 00:05:00 . (1/3)
5m
Xylene for 00:05:00 . (2/3)
5m
Xylene for 00:05:00 . (3/3)
5m
Alcohol absolute for 00:01:00 . (1/3)
1m
Alcohol absolute for 00:01:00 . (2/3)
1m
Alcohol absolute for 00:01:00 . (3/3)
1m
96% alcohol for 00:01:00 . (1/2)
1m
96% alcohol for 00:01:00 . (2/2)
1m
70% alcohol for 00:01:00 .
1m
Milli-Q water for 00:01:00 . (1/2)
1m
Milli-Q water for 00:01:00 . (2/2)
1m
Antigen retrieval
30m
HIER:
Place the slides in 10 millimolar (mM) Tris and 1 millimolar (mM) EDTA buffer 9 in the PT module.
Heat for 00:20:00 at 99 °C , total program lasts 01:10:00 .
20m
Wash 00:05:00 in Milli-Q and 00:05:00 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
Endogenous peroxidase block
15m
Immerse the slides in 0.9% H2O2 in TBS for 00:15:00 at Room temperature .
15m
Wash.
Wash 2x 3-5 minutes in TBS.
Wash 1x TBST.
Staining protocol: First 2 antibodies, detected with TSA reagents
3h 45m
Dry the slides around the specimen using lint-free tissue.
Apply 100-300 µL 10% NGS in TBST per slide, make sure the specimen is covered completely.
Incubate for 00:15:00 at Room temperature .
15m
Dilute the primary antibody or antibodies in 10% NGS in TBST.
Tap off the solution and apply 100-300 µL diluted primary antibody.
Incubate for 01:00:00 at Room temperature or Overnight at 4 °C (depending on the antibody).
1h
Wash 3x 3-5 min TBST.
Apply 100-300 µL 1:5 diluted (in TBST) Bright Vision goat anti mouse or rabbit HRP per slide.
Incubate 00:30:00 Room temperature .
30m
Wash 3x 3-5 min TBST.
Prepare the TSA-AF488 reagent just before use.
Pipet 100-300 µL reagent per slide and incubate 00:15:00 Room temperature .
15m
Wash 3x 3-5 min TBST.
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 10 millimolar (mM) Tris and 1 millimolar (mM) EDTA 9 in the PT module at 99 °C for 00:20:00 .
- 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 00:15:00 Room temperature .
15m
Wash 1x 3-5 minutes in TBS and 1x TBST.
Apply 100-300 µL 1:5 diluted (in TBST) Bright Vision goat anti mouse or rabbit HRP per slide.
Incubate 00:30:00 Room temperature .
30m
Wash 3x 3-5 min TBST.
Prepare the TSA-AF555 reagent just before use.
Pipet 100-300 µL reagent per slide and incubate 00:15:00 Room temperature .
15m
Wash 3x 3-5 min TBST and store at 4 °C Overnight if not proceeding immediately.
- After the second TSA reaction denature the previous antibody or antibodies by heating the slides in freshly prepared 10 millimolar (mM) Tris and 1 millimolar (mM) EDTA 9 in the PT module at 99 °C for 00:20:00 .
- Cool in MilliQ water 00:05:00 .
25m
Continue to AlexaFluor staining.
Staining protocol: AlexaFluor staining
45m
Dry the slides around the tissue.
Apply 100-300 µL 10% NGS in TBST per slide, make sure the tissue is covered completely.
Incubate 00:15:00 Room temperature .
15m
Dilute the primary antibodies in 10% NGS in TBST.
Tap off the blocking solution and apply 100-300 µL diluted primary antibodies.
Incubate 1-2h Room temperature or Overnight 4 °C .
Wash 3x 3-5 min TBST.
Dilute the secondary, AlexaFluor conjugated, antibodies in TBST, 1:300, add Dapi, final concentration 1.7 µL .
Apply 100-300 µL of the secondary reagent solution per slide and incubate 00:30:00 at Room temperature .
30m
Wash 3x 3-5min TBST and 1x 00:05:00 Milli-Q.
Dry slides at Room 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 Room temperature or 4 °C .
Staining protocol: Second to fourth round AlexaFluor stainings
45m
Wash the slides after the coverslips are soaked off in TBS.
Bleach the attached AF647 and AF750 by soaking the slides in TBS/NaOH/H2O2, 30-60 min Room temperature .
Rinse in 1x TBS and MilliQ.
- Denature the previous antibodies by heating the slides in freshly prepared 10 millimolar (mM) Tris and 1 millimolar (mM) EDTA 9 in the PT module at 99 °C for 00:20:00 .
- Cool in MilliQ-water 00:05:00 and wash in TBS 00:05:00 .
30m
Apply 100-300 µL 10% NGS in TBST per slide, make sure the tissue is covered completely.
Incubate 00:15:00 Room temperature .
15m
Dilute the primary antibodies in 10% NGS in TBST.
Tap off the blocking solution and apply 100-300 µL diluted primary antibodies.
Incubate 1-2h Room temperature or Overnight 4 °C .
Wash 3x 3-5min TBST.
Dilute the secondary, AlexaFluor conjugated, antibodies in TBST, 1:300, add Dapi, final concentration 3.4 µL (1:150).
Apply 100-300 µL of the secondary reagent solution per slide and incubate 30-45 minutes at Room temperature .
Wash 3x 3-5min TBST and 1x 00:05:00 Milli-Q.
Dry slides at Room 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 if more rounds are stained.