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counterstained with dapi (1 μg/ml)  (Thermo Fisher)
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Thermo Fisher counterstained with dapi (1 μg/ml)
(A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) <t>using</t> <t>antibodies</t> recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with <t>DAPI</t> (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.
Counterstained With Dapi (1 μg/Ml), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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counterstained with dapi (1 μg/ml) - by Bioz Stars, 2026-05
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dapi nuclear counterstain (1 μg/ml)  (Millipore)
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Millipore dapi nuclear counterstain (1 μg/ml)
(A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) <t>using</t> <t>antibodies</t> recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with <t>DAPI</t> (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.
Dapi Nuclear Counterstain (1 μg/Ml), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dapi nuclear counterstain (1 μg/ml) - by Bioz Stars, 2026-05
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dapi ij4′,6-diamidino-2-phenylindole) counterstain (1 μg ml−1)  (Kreatech Diagnostics)
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Kreatech Diagnostics dapi ij4′,6-diamidino-2-phenylindole) counterstain (1 μg ml−1)
(A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) <t>using</t> <t>antibodies</t> recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with <t>DAPI</t> (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.
Dapi ij4′,6 Diamidino 2 Phenylindole) Counterstain (1 μg Ml−1), supplied by Kreatech Diagnostics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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counterstain solution containing 1 μg/ 4,6-diamidino-2-phenylindole (dapi)  (Boehringer Mannheim)
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Boehringer Mannheim counterstain solution containing 1 μg/ 4,6-diamidino-2-phenylindole (dapi)
(A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) <t>using</t> <t>antibodies</t> recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with <t>DAPI</t> (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.
Counterstain Solution Containing 1 μg/ 4,6 Diamidino 2 Phenylindole (Dapi), supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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counterstain solution containing 1 μg/ 4,6-diamidino-2-phenylindole (dapi) - by Bioz Stars, 2026-05
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(A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) using antibodies recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with DAPI (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) using antibodies recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with DAPI (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.

Article Snippet: Then, sections were washed in PBST and incubated in species-specific Alexa Fluor 488- or 594- conjugated secondary antibodies (ThermoFisher Scientific, 1:500) for 1 hr at room temperature, counterstained with DAPI (1 μg/ml), mounted in Prolong Gold Antifade Mountant (ThermoFisher Scientific) and imaged using a Zeiss LSM700 confocal microscope equipped with a Plan-Apochromat 63x/1.40 objective and ZEN software (Carl Zeiss Microscopy).

Techniques: Expressing, Luciferase, Activity Assay, In Vitro, Western Blot, Immunolabeling, Labeling