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Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount (Vector Laboratories, Burlingame, California, United States) and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the R26R Cre reporter was not present."}

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Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount (Vector Laboratories, Burlingame, California, United States) and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the R26R Cre reporter was not present."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T12567","span":{"begin":0,"end":5},"obj":"UBERON:0002101"},{"id":"T12568","span":{"begin":30,"end":37},"obj":"NCBITaxon:33208"},{"id":"T12569","span":{"begin":152,"end":158},"obj":"UBERON:0000479"},{"id":"T12570","span":{"begin":199,"end":209},"obj":"GO:0008219"},{"id":"T12571","span":{"begin":225,"end":236},"obj":"CHEBI:31624"},{"id":"T12572","span":{"begin":335,"end":343},"obj":"CHEBI:53424"},{"id":"T12573","span":{"begin":349,"end":353},"obj":"NCBITaxon:9925"},{"id":"T12574","span":{"begin":354,"end":359},"obj":"UBERON:0001977"},{"id":"T12575","span":{"begin":416,"end":422},"obj":"NCBITaxon:9986"},{"id":"T12576","span":{"begin":428,"end":435},"obj":"CHEBI:32958"},{"id":"T12577","span":{"begin":436,"end":443},"obj":"CHEBI:15358"},{"id":"T12578","span":{"begin":436,"end":446},"obj":"PR:000027594"},{"id":"T12579","span":{"begin":447,"end":455},"obj":"GO:0042571"},{"id":"T12580","span":{"begin":463,"end":470},"obj":"GO:0007067"},{"id":"T12581","span":{"begin":561,"end":568},"obj":"GO:0007067"},{"id":"T12582","span":{"begin":570,"end":573},"obj":"CHEBI:37987"},{"id":"T12583","span":{"begin":587,"end":593},"obj":"NCBITaxon:9986"},{"id":"T12584","span":{"begin":604,"end":612},"obj":"GO:0042571"},{"id":"T12585","span":{"begin":636,"end":644},"obj":"GO:0042571"},{"id":"T12586","span":{"begin":646,"end":657},"obj":"GO:0005634"},{"id":"T12587","span":{"begin":676,"end":680},"obj":"CHEBI:51231"},{"id":"T12588","span":{"begin":840,"end":850},"obj":"UBERON:0001062"},{"id":"T12589","span":{"begin":904,"end":911},"obj":"GO:0005634"},{"id":"T12590","span":{"begin":940,"end":943},"obj":"CHEBI:37987"},{"id":"T12591","span":{"begin":952,"end":958},"obj":"GO:0005634"},{"id":"T12592","span":{"begin":1050,"end":1057},"obj":"NCBITaxon:33208"},{"id":"T12593","span":{"begin":1094,"end":1115},"obj":"_FRAGMENT"},{"id":"T12594","span":{"begin":1142,"end":1148},"obj":"UBERON:0003695"},{"id":"T12595","span":{"begin":1120,"end":1148},"obj":"UBERON:0003696"},{"id":"T12596","span":{"begin":1224,"end":1229},"obj":"UBERON:0004905"},{"id":"T12597","span":{"begin":1259,"end":1270},"obj":"UBERON:0006052"},{"id":"T12598","span":{"begin":1310,"end":1316},"obj":"_FRAGMENT"},{"id":"T12599","span":{"begin":1321,"end":1326},"obj":"UBERON:0009551"},{"id":"T12600","span":{"begin":1378,"end":1384},"obj":"UBERON:0000479"}],"relations":[{"id":"R8315","pred":"_lexicallyChainedTo","subj":"T12599","obj":"T12598"},{"id":"R8314","pred":"_lexicallyChainedTo","subj":"T12594","obj":"T12593"}],"text":"Limbs from mutant and control animals at E13.5 and E14.5 were dissected and frozen in OCT (Sakura Finetek,Torrence, CA, United States). Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount (Vector Laboratories, Burlingame, California, United States) and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the R26R Cre reporter was not present."}