Fluorescence Immunostaining of Cerebellar Organoid Slices

Abstract

Source: Silva, T. P., et al., Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining. J. Vis. Exp. (2020)

This video demonstrates the immunofluorescence staining of cerebellar organoid slices to identify specific target antigens expressed in various cerebellar neurons, which are indicative of organoid maturation.

Protocol

1. Immunostaining of organoid slices

1. Place the microscope slides containing organoid sections in a copling jar with 50 mL of prewarmed 1x PBS, holding up to 10 slides back-to-back.
   NOTE: All organoid sections should be submerged in liquid.
2. Incubate for 45 min at 37 °C to degelatinize slides.
3. Wash 1x with 50 mL of 1× PBS for 5 min at RT: Transfer the slides to a copling jar containing fresh 1× PBS.
4. Transfer the slides to a copling jar containing 50 mL of freshly prepared glycine (Table 1) and incubate for 10 min at RT.
5. Transfer the slides to a copling jar containing 50 mL of 0.1% triton (Table 1) and permeabilize for 10 min at RT.
6. Wash with 1× PBS for 5 min 2x.
7. Prepare the immunostaining dish with 3 mm paper soaked in 1× PBS. Dry the slides with tissue all around the slices and place them onto 3 mm paper. With a Pasteur pipette, cover the whole surface of the slides with a blocking solution (Table 1), 0.5 mL per slide. Incubate for 30 min at RT.
8. Remove the excess blocking solution and dry the slides with a tissue all around the slices. Place 50 µL of the primary antibody (Table 2) diluted in blocking solution over the sections and cover with the coverslips. Place the slices in a previously prepared immunostaining dish. Incubate overnight at 4 °C.
9. Transfer the slides to a copling jar with 50 mL of TBST (Table 1), let the coverslips fall, and wash with TBST for 5 min 3x.
10. Place 50 µL of the secondary antibody diluted in blocking solution over the sections and cover with the coverslips. Place the slices in the previously prepared immunostaining dish. Incubate for 30 min at RT, protected from light.
11. Transfer the slides to a copling jar again and wash with 50 mL of TBST for 5 min 3x.
12. Dry the slides with tissue all around the slices and place the slices in a previously prepared immunostaining dish. With a Pasteur pipette, add 0.5 mL of DAPI solution over the whole surface of the slides. Incubate for 5 min at RT.
13. Repeat step 1.9.
14. Carefully dry the slides with a tissue. Add 50 µL of mounting medium drop by drop along the slide and then carefully lower a coverslip onto each slide, slightly bending it to avoid bubbles.

Table 1: Solutions for preparation of organoids for cryosectioning and immunostaining. Listed are all the components and volumes used to prepare the solutions used in the preparation of organoids for cryosectioning and immunostaining.

Gelatin/Sucrose Final concentration: 7.5%/15% w/w	1. Weigh 15 g of sucrose and 7.5 g of gelatin in a sterile Schott Glass Bottle and mix well 2. Pre-warm the PBS 1x at 65 °C 3. Add pre-warmed PBS 1x to a final weight of 100 g and mix well 4. Place the Schott Glass Bottle in a heating plate at 65 °C and shake until the gelatin melts 5. Incubate at 37 °C until the solution stabilizes
Glycine Final concentration: 0.1 M	Add 0.37 g glycine to 50 mL of freshly prepared PBS 1x.
Triton solution Final concentration: 0.1 % w/v	1. Prepare a 10 % Triton X-100 stock: 5 g of Triton X-100 in 50 mL of PBS 1x 2. Add 0.5 mL of Triton X-100 stock to 50 mL of PBS 1x.
TBST 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05 % w/v Tween-20	20 mL Tris 1 M 30 mL NaCl 5 M 5 mL Tween-20 (10 % stock: 5 g of Tween-20 in 50 mL water) Fill to 1 L with water.
Blocking Solution	Add 5 mL of fetal bovine serum (FBS, final concentration: 10 % v/v) to 50 mL of TBST.
DAPI solution	Add 15 µL of DAPI stock solution (1 mg/mL) to 10 mL of distilled water
Mowiol	1. Add 2.4 g of Mowiol to 6 g of glycerol and shake for 1 h in a pre-warmed plate at 50 °C 2. Add 6 mL of distilled water and shake for 2 h 3. Add 12 mL of Tris 200 mM (pH 8.5) and shake for 10 min 4. Centrifuge at 5,000 × g for 15 min5. Aliquot and store at -20 °C.

Table 2: Primary antibodies. The primary antibodies, clone, and optimized dilutions used for immunostaining are listed.

Antibody	Host species	Dilution
BARHL1	rabbit	1/500
CALBINDIN	rabbit	1/500
MAP2	mouse	1/1000
N-CADHERIN	mouse	1/1000
NESTIN	mouse	1/400
OLIG2	rabbit	1/500
PAX6	rabbit	1/400
SOX2	mouse	1/200
TBR1	rabbit	1/200
TBR2	rabbit	1/200
TUJ1	mouse	1/1000

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3MM paper	WHA3030861	Merck
Anti-BARHL1 Antibody	HPA004809	Atlas Antibodies
Anti-Calbindin D-28k Antibody	CB28	Millipore
Anti-MAP2 Antibody	M4403	Sigma
Anti-N-Cadherin Antibody	610921	BD Transduction
Anti-NESTIN Antibody	MAB1259-SP	R&D
Anti-OLIG2 Antibody	MABN50	Millipore
Anti-PAX6 Antibody	PRB-278P	Covance
Anti-SOX2 Antibody	MAB2018	R&D
Anti-TBR1 Antibody	AB2261	Millipore
Anti-TBR2 Antibody	ab183991	Abcam
Anti-TUJ1 Antibody	801213	Biolegend
Coverslips 24x60mm	631-1575	VWR
DAPI	10236276001	Sigma
Fetal bovine serum	A3840001	ThermoFisher
Gelatin from bovine skin	G9391	Sigma
Glass Copling Jar	E94	ThermoFisher
Glycine	MB014001	NZYtech
Monothioglycerol	M6154	Sigma
Mowiol	475904	Millipore	Mounting medium
Triton X-100	9002-93-1	Sigma
Tween-20	P1379	Sigma
SuperFrost Microscope slides	12372098	ThermoFisher	Adhesion microscope slides