Jun 01, 2026
Immunofluorescence (IF) Protocol: Detection of GPNMB and TFE3 in Cultured ASPS Cell Lines
- Franz Zemp1,
- Lousia Guignard1,
- Douglas Mahoney1
- 1University of Calgary
Protocol Citation: Franz Zemp, Lousia Guignard, Douglas Mahoney 2026. Immunofluorescence (IF) Protocol: Detection of GPNMB and TFE3 in Cultured ASPS Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99eyqv3e/v1
Manuscript citation:
Zemp et al. 2026. GPNMB-directed CAR T-cell therapy against MiT/TFE family fusion-driven solid tumors. Nature Cancer. Accepted.
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: May 27, 2026
Last Modified: June 01, 2026
Protocol Integer ID: 318070
Keywords: tfe3 proteins in cultured alveolar soft part sarcoma, tfe3 in cultured asps cell lines purpose immunofluorescence, cultured alveolar soft part sarcoma, cultured asps cell lines purpose immunofluorescence, immunofluorescence, tfe3 protein, detection of gpnmb, simultaneous detection of gpnmb, cell line
Abstract
Purpose Immunofluorescence staining for the simultaneous detection of GPNMB and TFE3 proteins in cultured Alveolar Soft Part Sarcoma (ASPS) cell lines.
Guidelines
Notes
- Protect samples from light after addition of fluorescent secondary antibodies to preserve signal intensity.
- Include appropriate controls (single-color, isotype, and no-primary-antibody controls) in every experiment.
- Fixation and permeabilization times are not specified in the original protocol. Standard times are typically 10–20 min for fixation and 10–15 min for permeabilization at room temperature — these should be optimized for the specific cell line used.
- This protocol is designed for manual staining of cultured cells grown on slides or coverslips.
Materials
- Opti-MEM™ medium supplemented with 10% FBS
- 4% Paraformaldehyde (PFA) in PBS (fixative)
- 0.5% Triton X-100 in PBS (permeabilization solution)
- Goat polyclonal anti-human GPNMB antibody (R&D Systems, AF2550, 20 µg/mL)
- Rabbit monoclonal anti-TFE3 antibody (Sigma Cell Marque, 354R-15, 1:25)
- Alexa Fluor® 647 Donkey anti-Goat IgG (Thermo, cat. no. A-21447, 1 µg/mL) – for GPNMB detection
- DyLight™ 488 Donkey anti-rabbit IgG (BioLegend, cat. no. 406404, 1 µg/mL) – for TFE3 detection
- ProLong™ Gold antifade reagent with DAPI (Invitrogen, cat. no. P36935)
Procedure
Culture ASPS cell lines in Opti-MEM™ supplemented with 10% FBS for 48 hours on appropriate chamber slides or coverslips.
Fix the cells with 4% paraformaldehyde (PFA).
Permeabilize the fixed cells using 0.5% Triton X-100.
Incubate cells overnight at 4°C with both primary antibodies simultaneously:
Goat polyclonal anti-human GPNMB (R&D Systems, AF2550) at 20 µg/mL
Rabbit monoclonal anti-TFE3 (Sigma Cell Marque, 354R-15) at 1:25 dilution
Incubate with secondary antibodies at 1 µg/mL for 30 minutes at room temperature:
Alexa Fluor® 647 Donkey anti-Goat IgG for GPNMB detection
DyLight™ 488 Donkey anti-rabbit IgG for TFE3 detection
Mount the samples with one drop of ProLong™ Gold antifade reagent containing DAPI.
Digitalize the stained slides using the ECHO Revolve fluorescent microscope (ECHO, RVL-100-M) at 40× resolution.