Multiplex fluorescent immunostaining of thin, fixed mouse brain tissue sections to characterize human iPSC-derived cell xenografts
Benjamin Trist, Louise Cottle
Abstract
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.
Attachments
Steps
Day 1 (~3-4 hrs)
Pre-heat oven and Antigen Retrieval (AR) buffer to 70°C.
Label scintillation vials to match labels on section storage plates (mouse and section series IDs, name, date etc.).
Transfer sections into scintillation vials using a transfer pipette or fine paintbrush.
Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation.
Remove anti-freeze solution and perform 3x 0h 7m 0s washes in 1x PBS at Room temperature with gentle agitation (1/3).
Remove anti-freeze solution and perform 3x 0h 7m 0s washes in 1x PBS at Room temperature with gentle agitation (2/3).
Remove anti-freeze solution and perform 3x 0h 7m 0s washes in 1x PBS at Room temperature with gentle agitation (3/3).
Antigen retrieval (AR) - Day 1
Incubate sections in pre-heated AR buffer for 0h 30m 0s at 70°C.
After antigen retrieval, allow sections to cool for 0h 30m 0s before proceeding with staining.
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation (1/3).
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation (2/3).
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation (3/3).
Blocking step - Day 1
Incubate sections in blocking solution for 1h 0m 0s at Room temperature with gentle agitation.
Primary antibody step - Day 1
Incubate sections with desired primary antibody combinations (to characterise either cortical or ventral midbrain derived xenografts) diluted in blocking buffer 1h 0m 0s at 4°C with gentle agitation.
Day 2 (~4hrs)
Perform 3 x 7 min washes in 1x PBST with gentle agitation.
Perform 3 x 0h 7m 0s washes in 1x PBST with gentle agitation (1/3).
Perform 3 x 0h 7m 0s washes in 1x PBST with gentle agitation (2/3).
Perform 3 x 0h 7m 0s washes in 1x PBST with gentle agitation (3/3).
Secondary antibody step
Incubate sections in secondary antibodies, that match the species of chosen antibodies, diluted in blocking buffer for 2h 0m 0s at Room temperature. Incubate in a dark room or cover the vials with foil to protect the fluorophores from light.
Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light.
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation keeping vials protected from the light (1/3).
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation keeping vials protected from the light (2/3).
Perform 3x 0h 7m 0s washes in 1x PBST with gentle agitation keeping vials protected from the light (3/3).
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 1h 0m 0s- 2h 0m 0s.
Remove ProLong Diamond Antifade mountant from storage at 4°C and allow the reagent to equilibrate toRoom temperature before use.
Coverslip slides with ProLong Diamond Antifade mountant and allow to set at Room temperature for at least 24h 0m 0s in the dark before proceeding with microscopy.
Image sections using fluorescent microscopy for subsequent xenograft characterization.
Slides can be stored at 4°C or -20°C.
