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Page 295 Berber et al. J Transl Genet Genom 2021;5:292-303 https://dx.doi.org/10.20517/jtgg.2021.35

Method 3
Retinal organoids were differentiated as described in Method 2, with a single modification. On day 6, 1.5
[30]
nM BMP4 was added to the medium, as demonstrated by Capowski et al. .

Immunofluorescence
Immunocytochemistry
Cryosections (10 µm) of retinal organoids were washed three times in PBS for 5 min at room temperature
(RT) and blocked in 10% normal goat serum (Abcam, Cambridge, UK) and 0.3% Triton X (VWR,
Pennsylvania, USA) in PBS for 1 h at RT. Samples were incubated in primary antibodies in 2.5% normal
goat serum and 0.1% Triton X in PBS overnight (ON) at 8 °C. Samples were washed three times in PBS for 5
min at RT, and incubated in the secondary antibodies in 2.5% normal goat serum, 0.5% 4’,6-diamidino-2-
phenylindole (DAPI, Molecular Probes, Leiden, the Netherlands), and 0.1% Triton X in PBS ON at 8 °C.
The secondary antibody solutions were aliquoted and stored at -20 °C, to reduce batch effects. Samples were
washed three times in PBS for 5 min at room temperature and mounted in Dako fluorescence mounting
medium (Agilent, California, USA). Slides were dried ON before imaging.

Antibodies
The following primary antibody dilutions were used: AP2A 1:250 (ab108311, Abcam, Cambridge, UK),
BRN3A 1:250 (ab245230, Abcam, Cambridge, UK), CRX 1:1000 (H00001406-M02, Abnova, Taipeh,
Taiwan), OPN1SW 1:1000 (JH455, gift from Jeremy Nathans, Johns Hopkins University, Maryland, USA),
RCVRN 1:1000 (AB5585, Sigma, Missouri, USA), RHO1D4 1:1000 (gift from Robert S. Molday, University
of British Columbia, Vancouver, Canada), SNCG 1:500 (H00006623-M01, Abnova, Taipeh, Taiwan), and
VSX2 1:1000 (HPA003436, Sigma, Missouri, USA). The following secondary antibodies and dilutions were
used: Alexa Fluor® 488 goat anti-rabbit IgG 1:667 (Thermo Fisher Scientific, Waltham, USA) and Alexa
Fluor® 594 goat anti-mouse IgG 1:667 (Thermo Fisher Scientific, Waltham, USA). For samples stained with
PNA, Alexa Fluor® 594 goat anti-mouse IgG was replaced with 4% Alexa 594-conjugated peanut agglutinin
(Invitrogen, California, USA).

Analysis
Retinal organoid yield quantification
The number of retinal domains was manually counted on day 10 for Method 1 and day 23 for Methods 2
and 3. The mean from three independent differentiations was calculated. To test for significance, a one-way
ANOVA test, post hoc Tukey test, and Bonferroni correction for multiple comparisons were performed.

The number of retinal organoids was counted on day 63 for Methods 2 and 3, and the mean was calculated
from three independent differentiations. A Mann-Whitney U-test was performed to test for significance.

Microscopy and image analysis
All slides were imaged on a confocal microscope Olympus Fluoview FV3000. To confirm successful retinal
organoid differentiation and maturation, closeup images of retinal organoid sections were taken. To control
for regional variability, the quantification of the relative expression of retinal markers was performed from
whole cryosections. Three organoids were analyzed per method and each timepoint. The relative area of the
stain was calculated compared to the DAPI-positive area (for nuclear markers AP2A, BRN3A, CRX, and
SNCG) or the whole cryosection (for cytosolic markers RCVRN and RHO). The quantification was
[34]
performed in Fiji, an updated distribution of ImageJ . Macros were written for the quantification, which
[35]
converted the images to 16 bits, performed thresholding according to method in , and measured the
remaining area.