mouse monoclonal anti fgf8 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Self-formation of concentric zones of telencephalic and ocular tissues and directional retinal ganglion cell axons

doi: 10.1101/2023.03.22.533827

Figure Lengend Snippet: A scheme of the procedure. (B) A diagram of CONCEPT organoids showing concentric zones of the anterior ectodermal progenitors. See also Figs.1, 2, 3, 5, S1, S10. (C) Morphology of cysts at day 2 showing the epithelial structure indicated by the apical reporter TJ::GFP at the lumen. (D) Morphology of CONCEPT organoids at day 26. (E-G) Expression of telencephalon (Tel) marker Foxg1, neuroretinal (NR) markers Vsx2 and Pax6 in mouse eyes at E10-10.5. Rostral optic stalk (OS) connected the telencephalic vesicle to the optic cup. (H-O) FOXG1+ telencephalic progenitors, VSX2+ and/or PAX6+ retinal progenitors formed concentric zones in CONCEPT organoids. N > 5 experiments. (P-W) In CONCEPT organoids, morphogens FGF8, BMP4, and BMP7 mRNA expression started at early stages and subsequently formed circular gradients. N > 5 experiments. Scale bars, 100 µm (C, E, M, O, P, S, T), 200 µm (I, K), 500 µm (Q, U), 1 mm (D, H, J, L, N, R, V, W).

Article Snippet: These primary antibodies were used: FOXG1 (Abcam, ab18259, 1:500), TUBB3 (Covance MMS-435P, 1:1000), FGF8 (1:500, R&D MAB323), RBPMS (1:200, PhosphoSolution 1830-RBPMS), ISL1 (1:500, DSHB 40.2D6), SNCG (1:200, Abcam ab55424), PAX2 (Invitrogen, 716000, 1:200), alpha A crystallin (Santa Cruz sc 22743, 1:500, shown as CRYAA in figure panels), beta crystallin (Santa Cruz sc-22745, 1:100, shown as CRY B in figure panels), CNTN2 (DSHB 4D7, 1:100), ALDH1A3 (Invitrogen, PA529188, 1:500), VAX1/2 (Santa Cruz sc-98613, 1:200), PAX6 (1:500, Covance PRB-278P), POU4F2 (Santa Cruz, SC-6026, 1:200), SIX3 (1:500, Rockland), and VSX2 (1:500, Millipore AB9016).

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: Self-formation of concentric zones of telencephalic and ocular tissues and directional retinal ganglion cell axons

doi: 10.1101/2023.03.22.533827

Figure Lengend Snippet: PAX2, FGF9, and FGF8 are differentially expressed in cluster 2, the major component of PAX2+ optic-disc cells. (B) PAX2 mRNA expression in CONCEPT organoids on day 25. Two PAX2+ concentric zones corresponding to the optic stalk (OS) and optic disc (OD) are labeled. (C) Dual-color immunocytochemistry indicates the co-localization of FGF8 and PAX2 in the optic-disc zone of CONCEPT organoids on day 25. (D-E) TUBB3+ axons grew towards and then along the cells that expressed high levels of FGF8 (D) and FGF9 mRNA (E) in CONCEPT organoids on day 25. Immunocytochemistry of TUBB3 was performed after in situ hybridization. (F-K) After the inhibition of FGF signaling with FGFR inhibitor PD 161570 during days 17-24, the number of RGC somas drastically reduced, and directional axon growth of RGCs was nearly absent, whereas FGF8 protein expression largely remained. CNTN2 immunocytochemistry before (F, H) and after FGF8 immunocytochemistry (G, I, J, K) are shown. N = 3/3 experiments. Scale bar, 250 µm (B), 100 µm (C-E), 1 mm (F), 200 µm (K).

Article Snippet: These primary antibodies were used: FOXG1 (Abcam, ab18259, 1:500), TUBB3 (Covance MMS-435P, 1:1000), FGF8 (1:500, R&D MAB323), RBPMS (1:200, PhosphoSolution 1830-RBPMS), ISL1 (1:500, DSHB 40.2D6), SNCG (1:200, Abcam ab55424), PAX2 (Invitrogen, 716000, 1:200), alpha A crystallin (Santa Cruz sc 22743, 1:500, shown as CRYAA in figure panels), beta crystallin (Santa Cruz sc-22745, 1:100, shown as CRY B in figure panels), CNTN2 (DSHB 4D7, 1:100), ALDH1A3 (Invitrogen, PA529188, 1:500), VAX1/2 (Santa Cruz sc-98613, 1:200), PAX6 (1:500, Covance PRB-278P), POU4F2 (Santa Cruz, SC-6026, 1:200), SIX3 (1:500, Rockland), and VSX2 (1:500, Millipore AB9016).

Techniques: Expressing, Labeling, Immunocytochemistry, In Situ Hybridization, Inhibition

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Differential HS sulphation regulates [Fgf8] gradient formation with differential kinetics. (A) Fgf8 mRNA is located at the CSB angle (arrowhead). This image is also shown enlarged in Fig. 3 C. Sep, septum; Ctx, cortex; GE, ganglionic eminences. (B) Schematic of ex vivo explant culture with Fgf8-infused bead implanted at the CSB angle (arrowhead). (C) Fgf8 immunofluorescence (here shown as greyscale) illustrating concentric rings (yellow) used to quantify [Fgf8] at increasing distance (d) from the Fgf8-bead edge. (D-G) Representative images of Fgf8 immunofluorescence (red) in sections though cultured explants under different conditions and time-points indicated next to panels, asterisk marks bead centre. (H-K) [Fgf8] gradient up to 200 µm from the bead. Data for 1, 2, and 4 h time-points coloured black, red, and blue respectively with BSA control data coloured green, asterisks indicate significant difference between each particular condition with its corresponding WT (Kolmogorov–Smirnov test; P <0.05 following a Bonferroni correction for multiple pairwise comparisons). (L-O) Total Fgf8 level within 200 µm of the bead. Cross indicates significant differences between bracketed time-points within a culture condition indicating fluctuating amplitude through time, while asterisks indicate significant difference at a particular time-point between each particular condition and WT (two-way ANOVA followed by Holm-Sidak post hoc test; P <0.05). (P-S) Fitted curve of H-I in log 10 [Fgf8] vs. log 10 [d] . Each plot shows all data points and average line of best fit (solid line) with dotted line indicating extrapolation to log­ 10 [Fgf8] axis with numbers on indicating intercept and underlined numbers the slope. Number of explants analysed (N): WT , 8; Heparanase, 5; Hs2st −/− , 5; Hs6st1 −/− , 4. Scale bar in D applies to D-G: 100 µm. In H-S values are shown as mean±s.e.m.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Ex Vivo, Immunofluorescence, Cell Culture, Control

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Differential HS sulphation differentially regulate Erk response to Fgf8. (A-D) Representative images of pErk immunofluorescence in sections though cultured explants representing different conditions and time-points indicated on panels, asterisk marks bead centre. (E-H) [pErk] gradient formed at different time points up to 200 µm from the bead. Asterisks indicate significant difference between each particular condition with its corresponding WT [Kolmogorov–Smirnov test ( P <0.05) following a Bonferroni correction for multiple pairwise comparisons]. (I-L) [Fgf8]/[pErk] dose response curves. Number of explants analysed: WT , 8; Heparanase, 5; Hs2st −/− , 5; Hs6st1 −/− , 4. Data for 1, 2, and 4 h time-points coloured black, red, and blue respectively. Scale bar in D applies to A-D: 100 µm. In E-L values are shown as mean±s.e.m.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Immunofluorescence, Cell Culture

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Analysis of Fgf8/Erk signalling components in the CSB region of E14.5. WT , Hs2st −/− , and Hs6st1 −/− embryos show correlation to Hs2st and Hs6st1 action ex vivo . (A) Hs2st and (B) Hs6st1 expression visualized by LacZ staining of Hs2st LacZ/+ and Hs6st1 LacZ/+ sections, respectively. Cross marks an area with very low Hs6st1 LacZ expression, N =4. (C-E) Fgf8 mRNA expression. N =5. Note that the image in C is an enlargement of that shown in Fig. 1 A. (F-H) Fgf8 protein expression. N =3. (I-K) Higher magnification of VZ angle in F-H. (L) Quantification of Fgf8 fluorescent intensities in I-K. P values are as depicted on graph. Error bars indicate standard error of mean. (M) Dose response of WT , Hs2st −/− , Hs6st1 −/− tissue showing increased sensitivity to Fgf8 in Hs2st −/− while Hs6st1 −/− tissue have decreased sensitivity to Fgf8 when compared to WT tissue. (N,O) Expression of Fgfr1 protein. N =4 for WT , N =3 for Hs2st −/− and Hs6st1 −/− . (Q-S) pErk protein expression. N =3. (T-V) Higher magnification of ventricular zone in Q-S. In A, B, C-K and N-V arrowheads indicate apical surface of the ventricular zone at the CSB angle; arrows in H, P, S indicate Fgf8 High , Fgfr1 Low , pErk Low septal area in Hs6st1 −/− embryos. N =3. Ctx, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; Sep, septum. Scale bars: A applies also to B: 200 µm; C applies to C-E: 100 µm; F applies to F-H: 100 µm; I applies to I-K: 100 µm; N applies to N-P: 100 µm; and Q applies to Q-S: 100 µm; T applies to T-V: 100 µm.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Ex Vivo, Expressing, Staining