Immunofluorescence

Section I. Hazardous Reagent Safety & Preparation of key solutions (section is for both protocols)

Mandatory Safety Rules (read primary MSDS and review EH&S guidelines before proceeding)

• Fume Hood Requirement: All weighing and mixing of dry powders (PFA and NaN3) must be performed inside a certified chemical fume hood.
• Respiratory Protection: Per SDS guidelines, wear a well-fitted N95 mask when weighing powders to prevent inhalation of toxic dust (NaN3 can be fatal if inhaled; PFA is a carcinogen/irritant). Notify others in the room to stay out or wear a mask during this process.
• Chemical Incompatibility: Keep NaN3 away from acids (liberates fatal gas) and heavy metals (forms explosive azides).

• Disposal: Do not pour NaN3 or PFA down the drain. Dispose of all waste via your institution's hazardous waste guidelines.

   

Making 4% PFA Buffered in 1X PBS

1. In a fume hood, add 4g PFA powder into 90 mL MilliQ water.
2. Add 10 uL of 10N NaOH and shake gently.
3. Place the closed bottle in a 65°C water bath (or equivalent) for ~1 hour. Occasionally shake until dissolved.
4. Cool to RT and add 10 mL of 10X PBS.
5. Filter with a 0.22 μm bottle-top filter.

Preparing NaN3 Storage Buffer (0.02% NaN3, preservative for storage of free-floating tissue sections)

1. Stock (2% NaN3): Weigh 0.2g powder (N95 mask required) and dissolve in 10 mL 1X PBS.
2. Add 1 mL of 2% stock to 99 mL 1X PBS.

This preservative is used for the storage of free-floating sections.

A note on the technical differences between these protocols: The original protocol used Heat-Induced Epitope Retrieval with Citrate buffer to break methylene bridges introduced by fixation, whereas the alternative protocol uses a gentler Glycine quenching method. If the signal is weak in your hands with either method, you might need to optimize the incubation time for either the Citrate buffer or Glycine steps to improve epitope accessibility for antibody binding.

Protocol 1: Original eLife Protocol

Age Definitions:

2-week-old: Postnatal day 14 (P14) 

6-week-old: Postnatal day 42 (P42)

Section II. Procedural Steps Fixation and Anatomical Handling

1a. 2-week-old (P14): Dissect and rinse postmortem whole brains in 1X ice-cold PBS. Place the brain on a chilled cutting surface (e.g., a metal plate on ice). Using a sharp, single-edge blade, perform a single vertical cut along the midline (longitudinal fissure) to ensure a clean midsagittal separation. Drop-fix half-brains in PBS- buffered 4% PFA overnight at 4°C.

1b. 6-week-old (P42): Administer Buprenorphine 30 min prior to anesthesia. Perform transcardial perfusion using 10 mL ice-cold 1X PBS followed by 10 mL buffered 4% PFA. Post-fix brains in 4% PFA overnight at 4°C.

Cryoprotection and Freezing

2. Equilibration: Replace PFA with 30% sucrose and incubate at 4°C for 2 days (or until the brain sinks to the bottom of the tube, indicating full equilibration).

3. Embedding: Freeze in O.C.T. medium. Place half-brains flat side (midline) down in the receptacle, ensuring the tissue is flush with the surface before freezing on a dry ice block. Store at -80°C.

Tissue Sectioning and Collection

4. Cut 25 um sagittal sections on a Leica CM3050S cryostat (or equivalent) with the chamber at -20°C and the specimen head at -21°C.

5a. 2-week-old (P14) tissue: Mount sections directly onto slides and allow them to dry. Dried slides can be used immediately or stored in a freezer (-20°C or -80°C) until staining.

5b. 6-week-old (P42) tissue: Move sections with a paintbrush to a 24-well plate filled with 1X PBS + 0.02% Sodium Azide (NaN3). Store the 24-well plates in a 4°C fridge until staining.

Antigen Retrieval (2-week-old Tissue Only)

6a. 2-week-old (P14): If slides were stored in the freezer, allow them to come to Room Temperature (RT) before beginning the staining process.

7a. 2-week-old (P14): Submerge slides in Antigen Retrieval Buffer (10 mM Citrate pH 6.0, 0.05% Tween-20) in a 95°C water bath for 20 minutes.

8a. 2-week-old (P14): Allow the solution to cool to RT before proceeding.

Note: 6-week-old (P42) tissue sections do not require heat-induced antigen retrieval.

Immunostaining

9. Incubate in Blocking Buffer (2% Normal Goat Serum, 0.3% Triton X-100, in 1X PBS) for 1 hour at RT.

10. Incubate in Blocking Buffer with primary antibodies overnight at 4°C (see Table for dilutions).

11. Wash 4X in 1X TBST at RT for 5 minutes each.

12. Incubate overnight with secondary antibodies at 4°C (see Table for dilutions).

13. Wash 4X in 1X TBST at RT for 5 minutes each.

14. Stain with 2.5 ug/mL DAPI in 1X PBS for 10 min. Wash in 1X PBS.

15. Mount in VECTASHIELD HardSet (Vector H-1400). Let slides cure overnight at 4°C in the dark.

16. Once cured, seal the edges of the coverslip with clear nail polish to prevent evaporation and secure the mount. Once nail polish cures, slides are now ready to image (or store in the dark at 4°C)

Protocol 2: Rice University Lab Protocol (Unpublished Adaptation)

Adapted from PMID: 35534561; utilizes chemical quenching.

General Note: For mice that are 6 weeks or older, transcardial perfusion for fixation may be preferable. This protocol starts following the IACUC-approved euthanasia method and isolation of the intact whole brain.

Section II. Procedural Steps Drop-Fixation (P14 or Younger)

1. Rinse dissected postmortem whole brains several times in 1X ice-cold PBS.

2. Place the brain on a chilled cutting surface (e.g., a metal plate on top of ice in an ice bucket). Using a sharp, single-edge blade, perform a single vertical cut along the midline (longitudinal fissure), ensuring a clean separation into two symmetrical hemispheres (midsagittal plane).

3. Carefully submerge half-brains in freshly prepared buffered 4% PFA (buffered with 1X PBS). Incubate overnight at 4°C with gentle shaking on a platform shaker in the cold room.

Cryoprotection and Freezing Half-Brain Tissue (Days 2–4)

4. Replace the buffered 4% PFA solution with 30% sucrose–PBS at 4°C.

5. Incubate at 4°C with gentle shaking for 48 hours, or until the half-brains sink to the bottom of 50-mL conical tubes.

6. Embedding:

• i. On a flat, room-temperature surface, place an O.C.T.-compatible receptacle and add enough

  O.C.T. to cover the bottom. Label the receptacle to ensure accurate identification later.

• ii. Carefully move a half-brain and place it in the receptacle with the midline (flat side) facing the bottom, ensuring it is flush and flat to the surface (gently push down to be flat).
• iii. Fill the remaining space with O.C.T. to cover the tissue.
• iv. Place the receptacle onto a dry ice block until completely frozen.
• v. Store at -80°C until tissue sectioning.

Tissue Sectioning (Day 5 or Later)

7. Cut sagittal sections (15–35 μm) on a Leica 1900 cryostat (or equivalent) with the chamber at - 20°C and the specimen head at -21°C.

8. Move each section with a fine-tipped paintbrush to a 24-well plate filled with room-temperature 1X PBS containing 0.02% sodium azide (NaN3). Store sections in a 4°C fridge until staining.

Note: NaN3 helps prevent microbial growth and tissue contamination. Read MSDS, wear necessary PPE, and handle properly.

Staining (Day 5 or Later)

9. Prepare 24-well plates with RT 1X PBS (1 section/well for postnatal; 2–3 sections/well for prenatal).

10. Wash each section 3X for 5 minutes in RT 1X PBS. Use a platform shaker for gentle nutation. Be careful not to damage or lose sections when exchanging solutions.

11. Exchange PBS for 250mM Glycine in 1X PBS and incubate at RT for 5 minutes.

Note: Glycine effectively neutralizes reactive aldehyde groups from PFA fixation, serving as a mild form of antigen unmasking to reduce background and enhance antibody access.

12. Exchange glycine for blocking solution (10% donkey serum, 0.3% Triton X-100 in 1X PBS). Incubate at RT for 30 minutes.

13. Dilute primary antibody in 5% donkey serum, 0.3% Triton X-100 in 1X PBS. Incubate at 4°C overnight on a platform shaker with gentle shaking.

14. The next day, equilibrate Invitrogen Prolong Glass Antifade Mountant (P36980) to RT for 1 hour.

15. Wash sections 1X in 0.1% Triton X-100 in 1X PBS by exchanging the primary antibody solution.

16. Exchange with secondary antibody + DAPI solution (5% donkey serum, 0.3% Triton X-100 in 1X PBS +0.3 μg/mL DAPI). Incubate in the dark with gentle shaking for 1 hour at RT.

Note: You can mount in medium containing DAPI, or do a separate incubation with DAPI-PBS solution after the secondary; if separate, you must wash again after DAPI incubation.

17. Wash 3X with room-temperature 1X PBS.

18. Carefully mount sections onto glass slides by putting a small amount of 1X PBS on the slide. Gently spread out the section and slowly remove PBS while ensuring the section remains flat and in position.

19. Add RT mountant per instructions. Let slides cure overnight at RT in the dark.

20. Image or store at RT (or 4°C for long-term storage).

Note: Donkey serum can be replaced by other serums (e.g., fetal bovine serum, goat serum). If you use different types of serum, it is recommended that the host-species of the secondary antibodies is the same as the serum.