Apr 20, 2026

Multiplexed immunofluorescence and RNA-FISH

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm9zk9l3p/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
  • 3Duke University
Sarah van Veen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm9zk9l3p/v1
Protocol Citation: Sarah van Veen, Justin T Savage, Dolores Irala 2026. Multiplexed immunofluorescence and RNA-FISH . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9zk9l3p/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: January 13, 2025
Last Modified: April 26, 2026
Protocol  Integer ID: 118160
Keywords: ASAPCRN, RNA-FISH, ATP13A4, atp13a4 mrna in astrocyte, atp13a4 mrna, analysis of atp13a4 expression, astrocyte, atp13a4 expression, atp13a4 puncta, custom atp13a4 probe, positive astrocyte region, visual cortex at postnatal stage, confocal microscopy, brain sections from p7, multiplexed immunofluorescence, resolution confocal microscopy, egfp mice, visual cortex, brain section, p21 aldh1l1
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Abstract
This protocol describes a multiplexed immunofluorescence and RNA-FISH method to detect Atp13a4 mRNA in astrocytes of the visual cortex at postnatal stages. Brain sections from P7, P14, and P21 Aldh1L1-EGFP mice are immunostained with anti-GFP antibodies to label astrocytes and hybridized with custom Atp13a4 probes. Amplification is performed using hairpins specific to the probes and initiator-labeled antibodies. Sections are imaged using high-resolution confocal microscopy, and Atp13a4 puncta are quantified within GFP-positive astrocyte regions using a FIJI-based custom pipeline. This approach enables spatially resolved analysis of Atp13a4 expression during postnatal development.
Materials
  • Custom Atp13a4 probes (Molecular Instruments)
  • Buffers and reagents (Molecular Instruments)
  • Primary antibody: anti-GFP (Aves Labs, Cat# GFP-1020, RRID:AB_10000240) 
  • Secondary antibody: Initiator-labeled (specific for GFP detection)
  • Mounting medium
  • FIJI software with custom pipeline
  • Microscope: Olympus FV 3000
  • Other reagents:
  • 1× DPBS with 0.1% Tween20 (PBST)
  • paraformaldehyde (PFA; Electron Microscopy Sciences, Cat# 19210)
  • 5× SSCT (sodium chloride sodium citrate with 0.1% Tween20)
  • Probe wash buffer
  • Amplification buffer
  • Hairpins specific to initiator-labeled secondary antibody and Atp13a4 probes
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Section preparation
Prepare five 20 μm brain sections containing the visual cortex from P7, P14, and P21 Aldh1L1-EGFP mice.
Mount sections directly onto glass slides and store at -80 °C until use.

Blocking and primary antibody incubation
2h 5m
Thaw slides at room temperature for 00:05:00 to remove the cryoprotective solution.

5m
Wash sections three times with 1× PBST.
Block sections with antibody buffer for 01:00:00 at Room temperature .

1h
Incubate sections with anti-GFP primary antibody (1:1,000) Overnight at 4 °C in a humidified chamber.

1h
Secondary antibody incubation
1h
Wash sections with PBST.
Incubate sections with 100 µL initiator-labeled secondary antibody diluted in antibody buffer for 01:00:00 at Room temperature .

1h
Fixation and pre-hybridization wash
15m
Fix sections with 4% PFA for 00:10:00 .

10m
Wash sections with 5× sodium chloride sodium citrate containing 0.1% Tween20 (5× SSCT) for 00:05:00 .

5m
Probe hybridization
Incubate sections with 100 µL of 16 nM Atp13a4 probe solution Overnight at 37 °C in a humidified chamber.

Post-hybridization wash
Remove excess probes by washing sections in a mixture of probe wash buffer and 5× SSCT at 37 °C .

Gradually increase the concentration of 5× SSCT during washes, ending with a 100% 5× SSCT wash.
Hairpin amplification
1h 15m
Add 200 µL amplification buffer to each section and incubate at Room temperature for 00:30:00 .

30m
Mix snap-cooled hairpins specific to the initiator-labeled secondary antibody and Atp13a4 probe with amplification buffer to create a 60 mM hairpin solution.
Incubate sections with the hairpin solution Overnight at Room temperature .

45m
Final wash and mounting
45m
Wash sections with 5× SSCT for 00:45:00 to remove excess hairpins.

45m
Dry sections and mount with mounting medium.
Imaging
Image sections within two days using an Olympus FV 3000 microscope at high magnification (60× objective with 2× optical zoom).
Acquire z-stack images (1 μm step size, 6 μm total z-stack depth) for the visual cortex (layers L1, L2-3, L4, and L5).
Capture images from three sections per animal for three animals at each time point (e.g. P7, P14, and P21).
Data analysis
Process images using a FIJI custom pipeline.
Threshold and quantify Atp13a4 puncta inside and outside GFP-positive astrocyte areas.
Analyze data using software available at https://github.com/Eroglu-Lab/Irala_2024_image_analysis/blob/main/20230717_irala_fish_macro.ijm.