TITLE: Free floating immunofluorescent staining protocol on mouse brain sections
AUTHORS: Giselle Sagredo, YuHong Fu

[DESCRIPTION]
This protocol describes our multiplex fluorescent immunohistochemistry protocol used to identify pathological signatures in human iPSC-derived cells within thin, fixed mouse brain tissue section series’. 
We apply this workflow for post-mortem assessment of the inclusions within human iPSC-derived cells which have been transplanted into the living brain of athymic mice.

[GUIDELINES]
IMPORTANT: perform all antibody incubation steps and steps following in minimal light so as not to bleach signals prios to imaging

[STEPS]
SECTION: Day 1 - Tissue preparation
1. 30 um mouse brain sections were stored in anti-freeze solution at -20 °C until required. 
Remove samples from freezer and equilibrate at Room temperature for 10 min - 20 min
Pour sections into a well insert in a 6-well plate to separate storage solution from section
 Move the well insert to another well containing approximately 6 mL of 1x PBS. Wash at least 5x with 1x PBS for 5 min each on an orbital shaker using low speed at Room temperature
SECTION: Antigen retrieval
2. Incubate the sections in 10mM sodium citrate buffer (pH9.0) for 30 min. Let it cool to Room temperature
Rinse the sections 3x5 min each in 1X PBS
SECTION: Quenching aldehyde group
3. Weigh NaBH4 to make 0.1~0.5% in 1X PBS, made fresh
Move the insert with sections into the fresh-made solution for 30 min at Room temperature
Wash 2x 5 min in 1X PBS
SECTION: Blocking
4. Incubate sections in normal donkey serum IF blocking buffer120 min Room temperature on shaker 60 rpm
SECTION: Primary antibody incubation
5. Make primary antibody cocktails in blocking buffer
Prepare ~300 µL per sample of primary antibody solution consisting of selected primary
antibody (diluted appropriately) in home-made normal donkey serum IF blocking
buffer
Transfer sections from well insert into wells of black porcelain spot plate containing
primary antibody solution to bind to the antigen(s) of interest
Place the plate on a rotating mixer using low speed (speed 7 rpm) and incubate 4320 min at
4 °C (or 3X night/ over weekend)
SECTION: Day 2 - Secondary antibodies
6. The following day, pour sections into a well insert in a 6-well plate to separate
sections from primary antibody solution.
Wash sections 3 times with 1x PBST at Room temperature. Note: 30 s for the first two rinses, 3x 10 min for additional washing
Prepare 300 µL per sample of secondary antibody solution consisting of appropriate
secondary antibody + Hoechst 33342 (diluted accordingly) in blocking buffer (shield
solution from light)
Transfer sections into the black porcelain spot plate containing 300 µLsecondary
antibody cocktail
Incubate for 120 min at Room temperature on orbital shaker using low speed (shield solution from light).
Pour sections into a well-insert in a 6-well plate containing 1X PBST to separate sections from the secondary antibody solution
Continuing to shield samples from light, wash 3 times with 1x PBS for 5 min at Room temperature
SECTION: Mounting
7. Pour sections into a glass petri dish
Submerge a glass slide into the 1x PBS and use a fine paintbrush to coax the sections towards the slide
Gently tap the sections onto the slide, making sure there are no wrinkles or folds
Repeat until all sections are mounted onto the slide(s)
SECTION: Cover-slipping
8. After sections are dried onto the slide(s), about 15 min at Room temperature or until sections look opaque (remember to shield slides from light), apply an appropriate aqueous mounting medium
(hardening or non-hardening). Antifading (DAKO Fluorescence Mounting Medium
is preferred if using a fluorescent conjugated secondary antibody
Using tweezers, place a coverslip on top of the medium. Cover with filter paper and press down firmly to remove excess mounting medium
Image sections using an appropriate microscope. Store in a dark slide box at 4 °C