TITLE: Multiplex fluorescent immunostaining of thin, fixed mouse brain tissue sections to characterize human iPSC-derived cell xenografts
AUTHORS: Benjamin Trist, Louise Cottle

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

[STEPS]
SECTION: Day 1 (~3-4 hrs)
1. Pre-heat oven and Antigen Retrieval (AR) buffer to 70 °C.

SECTION: Day 1 (~3-4 hrs)
2. Label scintillation vials to match labels on section storage plates (mouse and section series IDs, name, date etc.).
SECTION: Day 1 (~3-4 hrs)
3. Transfer sections into scintillation vials using a transfer pipette or fine paintbrush.
SECTION: Day 1 (~3-4 hrs)
4. Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation.
 


SECTION: Antigen retrieval (AR) - Day 1
5. Incubate sections in pre-heated AR buffer for 30 min at 70 °C.

SECTION: Antigen retrieval (AR) - Day 1
6. After antigen retrieval, allow sections to cool for 30 min before proceeding with staining.

SECTION: Antigen retrieval (AR) - Day 1
7. Perform 3x  7 min washes in 1x PBST with gentle agitation.

SECTION: Blocking step - Day 1
8. Incubate sections in blocking solution for 60 min at Room temperature with gentle agitation.

SECTION: Primary antibody step - Day 1
9. Incubate sections with desired primary antibody combinations (to characterise either cortical or ventral midbrain derived xenografts) diluted in blocking buffer 60 min at 4 °C with gentle agitation.

SECTION: Day 2 (~4hrs)
10. Perform 3 x 7 min washes in 1x PBST with gentle agitation.

SECTION: Secondary antibody step
11. Incubate sections in secondary antibodies, that match the species of chosen antibodies, diluted in blocking buffer for 120 min at Room temperature. Incubate in a dark room or cover the vials with foil to protect the fluorophores from light. 

SECTION: Secondary antibody step
12. Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light. 

SECTION: Secondary antibody step
13. Mount tissue sections in a dimly lit room (to protect the fluorophores) onto super-frost slides pre-coated with gelatin-chrome alum and allow to dry in the dark at Room temperature for 60 min- 120 min.

SECTION: Secondary antibody step
14. Remove ProLong Diamond Antifade mountant from storage at 4 °C and allow the reagent to equilibrate toRoom temperature before use.

SECTION: Secondary antibody step
15. Coverslip slides with ProLong Diamond Antifade mountant and allow to set at Room temperature for at least 1440 min in the dark before proceeding with microscopy.

SECTION: Secondary antibody step
16. Image sections using fluorescent microscopy for subsequent xenograft characterization.
SECTION: Secondary antibody step
17. Slides can be stored at 4 °C or -20 °C.

SECTION: Day 1 (~3-4 hrs)
4.1. Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation (1/3).
SECTION: Day 1 (~3-4 hrs)
4.2. Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation (2/3).
SECTION: Day 1 (~3-4 hrs)
4.3. Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation (3/3).
SECTION: Antigen retrieval (AR) - Day 1
7.1. Perform 3x  7 min washes in 1x PBST with gentle agitation (1/3).

SECTION: Antigen retrieval (AR) - Day 1
7.3. Perform 3x  7 min washes in 1x PBST with gentle agitation (3/3).
SECTION: Antigen retrieval (AR) - Day 1
7.2. Perform 3x  7 min washes in 1x PBST with gentle agitation (2/3).
SECTION: Day 2 (~4hrs)
10.1. Perform 3 x 7 min washes in 1x PBST with gentle agitation (1/3).
SECTION: Day 2 (~4hrs)
10.2. Perform 3 x 7 min washes in 1x PBST with gentle agitation (2/3).
SECTION: Day 2 (~4hrs)
10.3. Perform 3 x 7 min washes in 1x PBST with gentle agitation (3/3).
SECTION: Secondary antibody step
12.1. Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light (1/3).
SECTION: Secondary antibody step
12.2. Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light (2/3).
SECTION: Secondary antibody step
12.3. Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light (3/3).