IF-HCR with whole mount Drosophila brains

This work is licensed under CC BY-SA 4.0

Process

This protocol is a variant of the base HCR RNA in situ hybridization protocol. It allows immunofluorescence and RNA-FISH in the same sample, extending experiment possibilities.

Before starting this protocol, be sure to get familiar with the base HCR RNA-FISH protocol.

If you find this protocol useful, please cite: https://doi.org/10.3389/fphys.2022.1051544

Day 1: dissection, permeablization, and primary antibody

total/hands-on time ~ 3hr/1hr

1. Prepare 4% paraformaldehyde (PFA) in 1xPBS for fixation by diluting the 16% PFA stock with 1xPBS. Each sample requires 500uL.
2. Add RVC to an aliquot of RNase-free 1xPBST stock (see Material) to a final concentration of 2mM. Each sample requires 3mL.
3. Clean glass spot plates with distilled water and 70% ethanol. Wipe the plates dry with paper towel.
4. Pipette 400uL of 4% PFA into one spot of the dish. Mark the dish and put on ice.
5. Dissect 10 brains within 30min. Transfer dissected brains into the dish on ice.
6. Nutate for 20min at room temperature for fixation. Fixation time shall be accurate to prevent overfixing.
7. Rinse the sample three times with 1xPBST+RVC, each 400uL.
8. Permeablize the sample by washing three times with 400uL 1xPBST+RVC. Each wash is 20min at room temperature with nutation.
9. Transfer the brains to a 1.5mL tube. Remove the wash as much as possible.
10. Add 300uL of antibody buffer (see Material). Incubate for 1hr at room temp for blocking.
11. Dilute primary antibody in fresh antibody buffer (minimum in 50uL/sample). Pipette to mix well and spin down.
12. Remove blocking solution and exchange into the primary antibody solution.
13. Incubate at 4C overnight for primary antibody binding.

Day 2: secondary antibody, post-IF fixation and HCR hybridization

total/hands-on time ~ 5hr/1.5hr

1. Add RVC to an aliquot of RNase-free 1xPBSTw stock (see Material, not PBST) to a final concentration of 2mM. Each sample requires 7mL.
2. Rinse three times with 1xPBSTw+RVC and then wash three times with 400uL 1xPBSTw+RVC. Each wash is 20min at room temp with nutation.
3. Dilute secondary antibody in fresh antibody buffer (minimum in 200uL/sample). Pipette to mix well and spin down.
4. Remove the last wash and exchange into secondary antibody solution.
5. Incubate at room temperature for two hours.
6. Perform the 1xPBSTw+RVC rinse-wash as described in Step#2 above. In the last 20min wash, use 1xPBSTw without RVC.
7. Prepare 4% PFA in 1xPBS and keep on ice until use.
8. Remove the last wash and add 400uL of 4% PFA for post-IF fixation. Incubate at room temp for 20min with nutation.
9. Perform the 1xPBSTw+RVC rinse-wash as described in Step#2. Change the three 20min washes into two 5min washes.
10. Remove the last wash and follow Day 2 of the base protocol for probe hybridization.

Day 3 - Day 4

Subsequent procedures are identical to the base protocol.

Material and Storage

For hybridization buffer and other FISH reagents, refer to the base protocol. Only reagents specific to this variant protocol are listed below.

Working solution

When preparing working solution, use 10% Triton X-100 and 10% Tween-20 stock solution.

1xPBST

1xPBS, 0.2% Triton X-100

1xPBSTw

1xPBS, 0.1% Tween-20

antibody buffer

commericial available from Molecular Instruments

antibody buffer (alternatives)

1xPBSTw, supplemented with 5% NGS or 1% BSA (w/v). Add RVC at a final concentration of 10mM.

Stock reagent

For Tween-20 and Triton X-100, prepare 10% (v/v) stock using ultrapure water which will aid pipetting.

Tested antibodies

Antibody ratios may or may not work for you and are dependent on experiment conditions. Below is a table of tested antibdies in my doctoral work lab:

Primary antibody

Secondary antibody