Sep 21, 2025

Public workspaceAn end-to-end workflow for GD2 immunofluorescence staining in unfixed human neuroblastoma tissue samples

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr84zolmk/v1
  • Sara Peggion1,
  • Clara Volz1,
  • Magdalena Trochimiuk1,
  • Isabelle Ariane Bley2,3,
  • Júlia Ramos4,
  • Konrad Reinshagen1,
  • Laia Pagerols Raluy1
  • 1Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany;
  • 2Division of Pediatric Stem Cell Transplantation and Immunology, Clinic of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany;
  • 3Research Institute Children’s Cancer Center Hamburg, 20251 Hamburg, Germany;
  • 4Institut Bonanova, Circumval.lació 8, 08003 Barcelona, Spain
Sara Peggion
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr84zolmk/v1
Protocol Citation: Sara Peggion, Clara Volz, Magdalena Trochimiuk, Isabelle Ariane Bley, Júlia Ramos, Konrad Reinshagen, Laia Pagerols Raluy 2025. An end-to-end workflow for GD2 immunofluorescence staining in unfixed human neuroblastoma tissue samples. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr84zolmk/v1
Manuscript citation:
To be added after peer review process.
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: In development
We are using this protocol, which is working, but we are also still developing and optimizing it.
Created: May 18, 2024
Last Modified: September 21, 2025
Protocol Integer ID: 100082
Keywords: Disialoganglioside, GD2, neuroblastoma, immunofluorescence, unfixed human neuroblastoma tissue samples this protocol, unfixed human neuroblastoma tissue sample, end workflow for gd2 immunofluorescence, gd2 immunofluorescence, staining of the membrane marker gd2, membrane marker gd2, immunostaining of ffpe tissue slice, unfixed tumor tissue sample, extracellular epitope loss, 2d fluorescence microscopy, ffpe tissue slice, initial tissue treatment, immunostaining
Abstract
This protocol allows the staining of the membrane marker GD2 directly in unfixed tumor tissue samples prior to 3D and/or 2D fluorescence microscopy.
By using this workflow it will be possible to avoid extracellular epitope loss (and consequent false negative results), which is experienced in the immunostaining of FFPE tissue slices due to initial tissue treatment.
Guidelines
The protocol was developed according to the Ordinance on Safety and Health Protection at Workplaces Involving Biological Agents (Biological Agents Ordinance - BioStoffV) of 15 July 2013 (Federal Law Gazette [BGBl.] Part I p. 2514), last amended by Article 1 of the Ordinance of 21 July 2021 (Federal Law Gazette I p. 3115).
Full text can be found at the following link:
https://www.gesetze-im-internet.de/englisch_biostoffv/englisch_biostoffv.html
Materials
Plastic/metallic ware
  • Pipette tips for battery-operated pipetting aid (5-10ml)
  • Lab spoons (potentially also stainless steel, autoclavable)
  • Magnetic stir bar(s)
  • Cryogenic tubes with screw cap (2ml are sufficient)
  • Petri plate (90x14,2mm)
  • Disposable scalpel
  • tweezers
  • Variable pipette tips (1-1000µL)
  • 96 well assay plate with black lateral walls and clear bottom with lid (e.g. Corning Incorporated - Ref. No. 3603)
  • Variable safe-lock tubes (1-50mL)
  • Variable brown safe-lock tubes (1-50mL)
  • Aluminium foil (e.g. LabSOLUTE, Cat.No. 7 696 857)

Glassware
  • Beaker (e.g. Pyrex‱ Griffin beakers, low form, with printed trace code)
  • Variable graduated laboratory bottle with cap, non-sterile, autoclavable

Equipment
  • Battery-operated pipetting aid
  • Precision balance
  • Magnetic stirrer
  • Research lab refrigerator (for 4°C storage)
  • Laboratory freezer (for -20°C storage)
  • Laboratory deep freezer (for -80°C storage)
  • Laboratory (fume) hood
  • Laboratory bench
  • Cryogenic liquid nitrogen container
  • Variable microliter pipettes (1-1000µL)
  • Mini-centrifuge
  • Vortex-mixer
  • (Digital) orbital shaker
  • (Portable digital) laboratory timer
  • Paraffin station

Reagents and buffers
  • Fetal Bovin Serum (e.g. Gibco‱ Fetal Bovine Serum, Ref. No. A5256801)
  • Bovine Serum Albumin (e.g. Sigma-Aldrich, Cat. No. A9418)
  • DPBS (Dulbecco's Phosphate Buffered Salin - Gibco‱, Ref. No. 14190-094)
  • Freezing medium for tissue specimens (Recovery‱ Cell Culture Freezing Medium, Gibco - Cat. No. 12648-10)
  • Purified Mouse anti-Human Disialoganglioside GD2 – Clone 14.G2a (BD Pharmingen‱, Cat. No. 554272) - 0,5mg/mL
  • Purified Mouse IgG2a, κ Isotype Control (BD Pharmingen‱, Cat. No. 556651) - 0,5mg/mL
  • Donkey Anti-Mouse IgG H&L conjugated with the dye Alexa Fluor‱ 647 (abcam, Cat. No. ab150107) - 2mg/mL
  • DAPI (e.g. Roth, Art. Nr. 6335.1)
  • Ultrapure distilled water (e.g. Sigma Aldrich, Cat.No. 38796-1L)
  • 4% formaldehyde with carbonate buffer/neutral pH (e.g. Engelbrecht, Cat.No. 10192)
  • Paraffin


Troubleshooting
Safety warnings
Use the laboratory hood according to manufacturer instructions in order to keep proper laboratory professionals' safety while working with biological material.
Ethics statement
This protocol was developed in agreement with the declaration of Helsinki on the use of human material for research. Ethical approval was obtained by the local ethics committee of Hamburg (No. 2020-10041-BO-ff). Make sure that written informed consent of all patients or their custodians is obtained in case you are going to use human tissue samples obtained during diagnostic biopsy, etc.
Experiments involving animals must be have obtained approval from an Institutional Animal Care and Use Committee or equivalent.
Before start
Ensure a lab coat, lab goggles (if necessary) and plastic gloves are worn throughout the whole protocol performance.

Preliminary steps
1h
2% FCS/1% BSA - washing buffer preparation:
Dissolve e.g. Amount2 mL FCS (Gibco‱ Fetal Bovine Serum, Ref. No. A5256801) with Amount1 g BSA (Bovine Serum Albumin - Sigma-Aldrich, Cat. No. A9418) in Amount98 mL DPBS (Dulbecco's Phosphate Buffered Salin - Gibco‱, Ref. No. 14190-094).

Mixing by using a magnetic stirrer is suggested; BSA has to be completely dissolved before usage.

Note
FCS has not to be sterile, but must be stored at 4°C. Otherwise it is possible to thaw FCS, which has been long-term stored at -20°C or -80°C.
BSA has not to be sterile either, but must be stored at 4°C.
Surplus washing buffer can be stored in a closed container at 4°C for up to 2 weeks.

The above mentioned amounts are merely a given example, the exact needed amounts should be calculated depending on tissue sample quantity and used assay plate; by using a 96 well assay plate the needed volume of washing buffer and further reagents can be minimized (Amount150 µL per well).


Mix
Tissue sample preparation:

Safety information
Throughout the staining protocol (possibly untested) tissue should be manipulated under laboratory hood conditions.

Note
Either fresh tissue (preserved in NaCl 0,9% solution upon collection) or thawed frozen tissue can be stained with this protocol. Tissue cryopreservation (in liquid nitrogen) should have been performed using freezing medium (e.g. Recovery‱ Cell Culture Freezing Medium, Gibco - Cat. No. 12648-10) for optimal tissue characteristic maintenance.

Specimens with max. Thikness3 mm thickness shall be prepared after tissue washing with DPBS (Amount500-1000 µL - depending on sample size) and placed separately in a 96 well assay plate with black lateral walls and clear bottom with lid (e.g. Corning Incorporated - Ref. No. 3603).
Dark lateral walls assure optimal protection against light exposure when incubation with a fluorochrome conjugated antibody is performed.

Following scheme proposes a possible staining design according to antibody type and patient's sample:
123
A
Pat. 1 - GD2
Pat. 1 - ISO
B
Pat. 2 - GD2
Pat. 2 - ISO
Following scheme proposes a completion of the previous staining design with adjunctive negative controls:
12345
A
Pat. X - GD2
Pat. X - ISO
Pat. X - 1st Ab only
Pat. X - 2nd Ab only
Pat. X - Dest. water only
6
A
Signal detection after tissue samples' incubation with only either primary or secondary antibody as well as only with ultrapure distilled water (e.g. Sigma Aldrich, Cat.No. 38796-1L) undermines staining results's reliability.
Critical
Toxic
Antibody dilution:
Primary antibodies - stored at 4°C, according to manufacturer's instructions
  • Purified Mouse anti-Human Disialoganglioside GD2 – Clone 14.G2a (BD Pharmingen‱, Cat. No. 554272) - Concentration0,5 mg/mL
  • Purified Mouse IgG2a, κ Isotype Control (BD Pharmingen‱, Cat. No. 556651) - Concentration0,5 mg/mL

Secondary antibody - stored at -20°C, according to manufacturer's instructions
  • Donkey Anti-Mouse IgG H&L conjugated with the dye Alexa Fluor‱ 647 (abcam, Cat. No. ab150107) - Concentration2 mg/mL

Temperature
Primary antibody dilution:
Note
After titration tests the optimum antibody concentration with strong positive signal and minimum background or nonspecific reactions was shown to be Concentration0,0025 mg/mL , which means a 1/200 dilution starting from the above mentioned antibody concentration.
Prepare the appropriate volume of DPBS (consider the example below) in a safe-lock tube (e.g. Eppendorf Safe-Lock Tubes).
Spin down the undiluted primary antibody for up to 2 seconds before pipetting it into DPBS. Pipette the right amount of the selected primary antibody into the appropriate volume of DPBS. Vortex the obtained diluted antibody solution for up to 2 seconds.
Repeat these steps in order to analogously dilute the isotype control antibody.
Store diluted antibody solutions at 4°C until usage.

Practical dilution example:
4 wells x 150µL/well = 600µL (tot. vol.) -> 3µL antibody + 597µL DPBS
Pipetting
Secondary antibody dilution:
Note
After titration tests the optimum antibody concentration with strong positive signal and minimum background or nonspecific reactions was shown to be Concentration0,01 mg/mL , which means a 1/200 dilution starting from the above mentioned antibody concentration.

Let the antibody thaw at room temperature.
Prepare the appropriate volume of DPBS in a brown safe-lock tube (e.g. Eppendorf Safe-Lock Tubes - brown), in order to avoid photobleaching.
Spin down the thawed undiluted secondary antibody for up to 2 seconds before pipetting it into DPBS. Pipette the right amount of the selected secondary antibody into the appropriate volume of DPBS. Vortex the obtained diluted antibody solution for up to 2 seconds.
Store diluted antibody solutions at 4°C until usage.

Practical dilution example:
4 wells x 150µL/well = 600µL (tot. vol.) -> 3µL antibody + 597µL DPBS
Pipetting
DAPI solution (4′,6-diamidino-2-phenylindole) preparation
Dilute DAPI (e.g. Roth, Art. Nr. 6335.1) in ultrapure distilled water (e.g. Sigma-Aldrich, Cat. No. 102539811) in order to reach a concentration of Concentration1 µg/mL by pipetting up and down in a brown safe-lock tube (e.g. Eppendorf Safe-Lock Tubes), in order to avoid photobleaching. Subsequently, vortex the tube containing DAPI diluted solution for up to 2 seconds.

Note
Undiluted DAPI must be stored at -20°C and thawed at room temperature upon dilution.
Surplus DAPI diluted solution can be stored in a closed container at 4°C for up to 6 months (avoiding exposure to light sources).


Pipetting
Mix
Tissue staining
5m

Note
Following steps require a a 200µL-pipette and appropriate tips.

1st washing step
Add Amount150 µL of the previously mixed washing buffer (Go to ) to each well containing a tissue specimen. Cover the 96 well assay plate. Complete this first washing step by placing the assay plate on an orbital shaker at Shaker20-30 rpm, Room temperature for 5 minutes.

Pipetting
Wash
2nd washing step
Discard the washing buffer. Repeat step 5 (Go to ).
Safety information
Discard washing buffer appropriately.

8m
Pipetting
Wash
Protein blocking
Discard the washing buffer. Add Amount150 µL of the previously mixed washing buffer (Go to ) to each well containing the tissue specimens and incubate them on an orbital shaker at Shaker20-30 rpm, Room temperature for 20 minutes, after covering the 96 well assay plate.

Note
Before any antibody application it is necessary to perform adequate protein blocking on tissue samples to prevent antibodies from binding to non-target epitopes.
Citation
Kyuseok Im, Sergey Mareninov, M. Fernando Palma Diaz, and William H. Yong (2020). An introduction to Performing Immunofluorescence Staining. Methods Mol Biol..
doi: 10.1007/978-1-4939-8935-5_26
LINK


20m
Pipetting
Wash
Critical
Incubation with the primary antibody
Discard the washing buffer.
Incubate each target tissue specimen with Amount150 µL of the previously diluted primary antibody (Go to ) on an orbital shaker at Shaker20-30 rpm for 4 hours at room temperature.

Note
For each human sample undergoing the staining protocol two specimens should be obtained. One specimen is incubated with diluted Purified Mouse anti-Human Disialoganglioside GD2 – Clone 14.G2a (BD Pharmingen‱, Cat. No. 554272); the second one serves as an appropriate negative control by being incubated with diluted Purified Mouse IgG2a, κ Isotype Control (BD Pharmingen‱, Cat. No. 556651), as displayed in the following Well Plate Map.

123
A
Pat. 1 - GD2
Pat. 1 - ISO
B
Pat. 2 - GD2
Pat. 2 - ISO


Incubation
Pipetting
3rd washing step
Discard the primary antibody solution. Add Amount150 µL of the previously mixed washing buffer (Go to ) to each well containing a tissue specimen. Cover the 96 well assay plate. Incubate on an orbital shaker (Shaker20-30 rpm, Room temperature ) for 5 minutes.

Pipetting
Wash
4th washing step
Discard the washing buffer. Repeat step 9 (Go to ).

Pipetting
Wash
Incubation with the secondary antibody
Discard the washing buffer.
Incubate each target tissue specimen with Amount150 µL of the previously diluted secondary antibody (Go to ) on an orbital shaker at Shaker20-30 rpm for 90 minutes at room temperature.

Note
From now on it is mandatory to protect the plate from light sources by wrapping it with aluminium foil (e.g. LabSOLUTE, Cat.No. 7 696 857).


Incubation
Pipetting
Critical
5th washing step
Discard the secondary antibody solution. Add Amount150 µL of the previously mixed washing buffer to each well containing a tissue specimen. Cover the 96 well assay plate. Incubate on an orbital shaker (Shaker20-30 rpm, Room temperature ) for 10 minutes.
Do not forget to keep the assay plate protected from light sources by wrapping it with aluminium foil.

Pipetting
Wash
6th - 7th washing step
Discard the washing buffer. Repeat step 12 twice (Go to ).

Note
These washing steps ( Duration00:10:00 repeated for three times) reduce unspecific secondary antibody binding at a minimum and therefore downscale unintended background signal.

Pipetting
Wash
Nuclei staining with DAPI (4′,6-diamidino-2-phenylindole)
Discard the washing buffer. Add Amount150 µL of the previously diluted DAPI solution (Go to ) to each well containing a tissue specimen. Cover the assay plate. Complete incubation by placing the assay plate on an orbital shaker at Shaker20-30 rpm, Room temperature for 30 minutes.

Incubation
Pipetting
Critical
9th washing step
Discard DAPI solution and wash tissue specimens by adding Amount150 µL of DPBS to each well. Cover the assay plate and incubate the samples on an orbital shaker at Shaker20-30 rpm, Room temperature for 5 minutes.

Safety information
Discard DAPI solution appropriately.

Pipetting
Wash
10th washing step
Discard DPBS and wash tissue specimens by adding Amount150 µL of ultrapure distilled water (e.g. Sigma Aldrich, Cat.No. 38796-1L) to each well. Cover the assay plate and incubate the samples on an orbital shaker at Shaker20-30 rpm, Room temperature for 5 minutes.
Pipetting
Wash
Tissue fixation
Discard distilled water. Add Amount150 µL of 4% formaldehyde with carbonate buffer/neutral pH (e.g. Engelbrecht, Cat.No. 10192) to each well containing a tissue specimen. Cover the assay plate. Formaldehyde fixation (at RT) over 1 hour is sufficient for thin tissue specimens (max. Thikness3 mm ) . In case of thicker tissue specimens, fixation may be prolonged by storing the samples at 4°C overnight.

Note
Formaldehyde fixed tissue can directly undergo confocal fluorescence microscopy in order to obtain 3D images and screen rapidly for GD2 expression pattern. Since most laboratories do not have direct access to a confocal microscope, tissue can be further paraffin embedded. FFPE tissue slices can be analyzed by mean of standard 2D fluorescence microscopy.
Detailed information about these further procedures can be found in: Peggion, S.; Volz, C.; Trochimiuk, M.; Bley, I.A.; Ramos, J.; Reinshagen, K.; Pagerols Raluy, L. - A Backwards Approach to GD2 Immunofluorescence in Human Neuroblastoma Tissue Samples: From Staining to Slicing. Cells 2025, 14, 1462. https://doi.org/10.3390/cells14181462


Pipetting
Toxic
Citations
Step 7
Kyuseok Im, Sergey Mareninov, M. Fernando Palma Diaz, and William H. Yong . An introduction to Performing Immunofluorescence Staining.
doi: 10.1007/978-1-4939-8935-5_26
Acknowledgements
The authors gratefully acknowledge the technical support from Dr. Antonio Virgilio Failla, Core Manager of the Microscopy Imaging Facility at the University Medical Centre Hamburg-Eppendorf.