Immunostaining of Neurons in Mouse Brain Slices Following Fluorescence In Situ Hybridization

Abstract

Source: Baleriola, J., et. al., Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization. J. Vis. Exp. (2015)

This video demonstrates the detection of mRNA within cholinergic axons in fixed mouse hippocampal slices using fluorescent in situ hybridization and antibody staining. Fluorescence microscopy reveals the spatial localization of probe-tagged mRNAs and choline acetyltransferase in cholinergic neurons.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. FISH assay followed by immunohistochemistry using heat-induced antigen/RNA unmasking
   1. Before starting, prepare all required material:
      1. Heat the hybridization oven to 40 ℃ and place a tray containing distilled water in the oven to create a humidified environment.
      2. Take a slide box and place paper towels soaked in distilled water inside to humidify it. Then, place the slide box in the hybridization oven.
      3. Remove the probes (both negative and target probes) from the fridge and heat them in the hybridization oven for 10 minutes. Then, let the probes cool down at RT.
      4. Equilibrate amplification reagents AMP 1-4 (included in the Fluorescent Multiplex Reagent Kit) at RT.
      5. Prepare antigen retrieval solution: 10 mM sodium citrate (pH 6), 0.05% Tween 20. Note: Solution can be stored at RT indefinitely and be used for future staining.
      6. Prepare 1x wash buffer diluting the 50x stock solution (in the Fluorescent Multiplex Reagent Kit) in RNase-free or DEPC-treated water.
         NOTE: 1x wash buffer can be stored at RT for 1 month and be used for future staining. 50x wash buffer might precipitate. If so, heat 50x wash buffer at 40 ℃ for 10 min before preparing the 1x solution.
   2. Remove slides from 100% ethanol and air-dry for 5 min at RT.
   3. Prepare a coplin jar containing 50 ml of antigen retrieval solution. Immerse slides in antigen retrieval solution and boil them for 10 min in a microwave oven.
      NOTE: Alternatively place slides horizontally in a 100 mm diameter round glass dish containing 100 ml of retrieval solution.
      1. Fill up a 1 L beaker with distilled water and place it in the microwave.
         NOTE: The water will “buffer” the heat when performing unmasking.
      2. Place the slides in antigen retrieval solution inside the microwave. Boil slides at high power for 5 min. Immediately after samples have stopped boiling, heat slides again at high power for an extra 5 min.
      3. Cool down slides at RT.
         NOTE: CRITICAL STEP: boiling duration and procedure should be determined by the user, depending on the microwave characteristics. The faster the boiling starts, the higher the chances of solution evaporation. In this case it is recommended that samples are boiled for shorter time periods more than twice to complete a total unmasking time of 10 min. On the contrary, if heating is performed at medium or low power, unmasking can be performed once for 10 min. However, if samples do not boil continuously for a total of approximately 10 min, this might result in partial unmasking of both the target RNA and protein to be detected.
   4. Wash slides three times with 1x PBS.
   5. Add 2 - 3 drops (~100 μl) of the dapB probe set to those brain slices used to assess background staining. NOTE: The dapB probe set targets a bacterial gene and should not recognize any mammalian RNA.
   6. Add 2 - 3 drops of the targeting probe set to those brain slices used to detect the RNA of interest. NOTE: a probe set targeting residues 20 - 1,381 of the mouse Atf4 mRNA is used in this example.
   7. Cover slides with parafilm to avoid probe evaporation, place them in the humidified slide box inside the hybridization oven, and incubate them at 40 ℃ for 2 hrs.
      ​NOTE: OPTIONAL: additional brain slices can be hybridized with a probe set targeting mouse Polr2A and used as positive controls.
   8. Wash slides twice with 1x wash buffer for 2 min at RT.
   9. Add 2 - 3 drops of amplification reagent AMP 1-FL to each brain slice, cover slides with parafilm, place them in the slide box, and incubate at 40 ℃ for 30 min in the hybridization oven.
   10. Wash slides twice with 1x wash buffer for 2 min at RT.
   11. Add 2 - 3 drops of amplification reagent AMP 2-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 ℃ for 15 min in the hybridization oven.
   12. Wash slides twice with 1x wash buffer for 2 min at RT.
   13. Add 2 - 3 drops of amplification reagent AMP 3-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 ℃ for 30 min in the hybridization oven.
   14. Wash slides twice with 1x wash buffer for 2 min at RT.
   15. Add 2 - 3 drops of amplification reagent AMP 4-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 ℃ for 15 min in the hybridization oven.
   16. Wash slides twice with 1x wash buffer for 2 min at RT and twice with 1x PBS.
   17. Add 100 - 200 μl (or enough volume to completely cover slices) of a blocking solution containing 3 mg/ml BSA, 100 mM glycine, and 0.25% Triton X-100 in PBS (pH 7.4) to the slides, cover them with parafilm, and incubate for 30 min at RT.
   18. Add 100 - 200 μl of anti-ChAT antibody diluted in blocking solution (1/100) to the slides, cover them with parafilm, and incubate for 2 days at 4 ℃. Make sure that brain slices don’t dry out. If needed, re-apply the anti-ChAT antibody solution to brain slices 24 hrs after incubation.
   19. Wash slides three times in 1x PBS for 5 min at RT.
   20. Add 100 - 200 μl of the appropriate Alexa-conjugated secondary antibody (e.g., Alexa-594 donkey anti-goat for this particular example) to the slides, cover with parafilm, and incubate for 1 hr at RT.
   21. Wash slides three times in 1x PBS for 5 min at RT.
   22. Wash slides once with distilled water.
   23. Mount slides with a DAPI-containing mounting medium.
   24. Visualize brain slices under a fluorescence microscope.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Custom probe targeting residues 20-1381 of the mouse Atf4 mRNA (NM_009716)	Advanced Cell Diagnostics	-	Probe
Custom probe targeting residues 15-1256 of the human ATF4 mRNA (NM_001675.2)	Advanced Cell Diagnostics	-	Probe
Negative control probe-DapB	Advanced Cell Diagnostics	310043	Probe
Positive control probe-mouse Polr2A (optional)	Advanced Cell Diagnostics	312471	Probe
Goat polyclonal anti-ChAT antibody	Millipore	AB144P
ProLong Gold mounting medium with DAPI	Life Technologies	P36935
RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)	Advanced Cell Diagnostics	320850	In situ hybridization kit