Journal: Experimental eye research

Article Title: Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina

doi: 10.1016/j.exer.2016.11.013

Figure Lengend Snippet: A) Typical in vitro differentiated retinal organoids with phase bright tissue. Apical surface is marked in red while green demarcates the basal side of the retinal epithelium. Scale bar: 500 μm. B) Immunostaining of cryosectioned retinal organoids demonstrating that the RPCs (VSX2+ PAX6+ Ki67+) differentiated from hESCs undergo mitosis (PH3) at the apical surface (ZO-1) scale bar: 100 μm. C) RPCs give rise to photoreceptors (CRX), horizontal (PROX1), amacrine (AP2α), and bipolar (bright VSX2 day 140, but not day 57) cells. Insets show bright VSX2 signal that co-stains with DAPI while asterisks (*) indicate bright artifacts. Non-specific red (VSX2 day 57) staining is outlined and marked with an asterisk. Scale bar: 100 μm.

Article Snippet: Sections were incubated with primary antibodies (see Table S1 ) diluted in blocking buffer for 2 hr at RT or overnight at 4°C, washed 3 X 5 min in TBST, incubated with secon dary antibodies diluted in blocking buffer for 1 hr at RT in the dark, washed 3 X 5 min in TBST with DAPI (1:1000) included in the last wash, and cover-slipped using Mowial mounting medium plus DABCO or ProLong Diamond (Thermo Fisher Scientific).

Techniques: In Vitro, Immunostaining, Staining

Journal: Experimental eye research

Article Title: Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina

doi: 10.1016/j.exer.2016.11.013

Figure Lengend Snippet: A) Immunostaining time course demonstrating RGC production in retinal organoid culture at the indicated day following initiation of differentiation from hESCs. The NBL resides roughly between the dotted lines and the basal layer is below. Scale bar: 100 μm. B) Quantitation of BRN3 immunofluorescent signal relative to DAPI signal from images similar to those shown in A. Data is the mean signal (see methods) from n images from m distinct preps. (n,m) is shown next for each point. Error is standard deviation. C) Tau immunostain demarcates axon outgrowth. Inset is magnified to the right. Scale bars: 100 μm, 25 um. D) Phase contrast images demonstrating axon outgrowth from a day 51 retinal organoid placed on Matrigel, but not from day 23 or 114 retinal organoids, prior to RGC genesis or after RGC death, respectively. Scale bar: 500 μm. Axon branching and growth cones at day 51 are shown at higher magnification. Scale bar: 100 μm.

Article Snippet: Sections were incubated with primary antibodies (see Table S1 ) diluted in blocking buffer for 2 hr at RT or overnight at 4°C, washed 3 X 5 min in TBST, incubated with secon dary antibodies diluted in blocking buffer for 1 hr at RT in the dark, washed 3 X 5 min in TBST with DAPI (1:1000) included in the last wash, and cover-slipped using Mowial mounting medium plus DABCO or ProLong Diamond (Thermo Fisher Scientific).

Techniques: Immunostaining, Quantitation Assay, Standard Deviation

Journal: Experimental eye research

Article Title: Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina

doi: 10.1016/j.exer.2016.11.013

Figure Lengend Snippet: A) Developmental time course of hESC-derived retina immunostained with anti-CD171-PE (Life Technologies) and anti-CD184-PE. DAPI is blue. With the CD184 antibody areas of non-specific staining or probable non specificity are outlined and marked with an asterisk. An independent CD171 antibody showed similar staining pattern (Figure S4). Scale bar: 100 μm. B) Flow cytometry plots of dissociated retinal organoid cells of the indicated age labeled with anti-CD184-APC and anti-CD171-PE. Each plot shows data comprised of cells from six dissociated retinal organoids. Quadrant lines drawn based on unstained cell or IgG control signal (both exhibited equivalent signal), while boxes gate the CD184hi or CD184hi CD171+ populations with percent of total cells shown within. CD184hi is discriminated from CD184+ based on a visual assessment of the minor population exhibiting higher levels of CD184. C) Immunostaining of pf weeks 11.5 and 13 human fetal retina. Noncontiguous pictures show the progression from nasal to temporal retina with cartoon showing approximate picture location in the retina. Insets show either cells from the GCL(a) or NBL(b) at 3.5X ONH (optic nerve head). At pf 13 inset b includes part of the emerging ONL as well. Staining (including that in Figure S5 which shows BRN3 detection for reference) was performed on adjacent retina sections, each stained with different antibodies. For each antibody all pictures were taken from the same section with identical exposure times. The only exception is the pf 11.5 central picture (temporal to the ONH) because the ONH was not found in the same section as the most central retina stained. Scale bar: 250 μm.

Article Snippet: Sections were incubated with primary antibodies (see Table S1 ) diluted in blocking buffer for 2 hr at RT or overnight at 4°C, washed 3 X 5 min in TBST, incubated with secon dary antibodies diluted in blocking buffer for 1 hr at RT in the dark, washed 3 X 5 min in TBST with DAPI (1:1000) included in the last wash, and cover-slipped using Mowial mounting medium plus DABCO or ProLong Diamond (Thermo Fisher Scientific).

Techniques: Derivative Assay, Staining, Flow Cytometry, Labeling, Control, Immunostaining