Histological Analysis of Limb Development

Methods used were as described previously (Tsang et al., 2007 (link)). In brief, limbs were fixed in 4% PFA, followed by demineralization in 0.5M EDTA (pH 8.0) before embedding in paraffin. Slides were stained with Alcian Blue for cartilage matrix and Fast Red for nuclei. Immunofluorescence was performed using antibodies against ATF4 (sc-200, Santa Cruz), ATF3 (HPA001562, Sigma), CHOP (sc-575, Santa Cruz), PPP1R15A (sc-825, Santa Cruz), FGF21 (42189, AIS) and Sox9 (AB5535, Millipore).

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Wang C., Tan Z., Niu B., Tsang K.Y., Tai A., Chan W.C., Lo R.L., Leung K.K., Dung N.W., Itoh N., Zhang M.Q., Chan D, & Cheah K.S. (2018). Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia. eLife, 7, e37673.

Publication 2018

sc-200 HPA001562 sc-575 AB5535

Alcian blue Antibodies Atf4 Cartilage Chop Edta Fgf21 Immunofluorescence Red nuclei Sox9

Corresponding Organization :

Other organizations : University of Hong Kong, Kyoto Pharmaceutical University, The University of Texas at Dallas, Center for Systems Biology, Tsinghua University

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Variable analysis

independent variables

Limb fixation in 4% PFA
Demineralization in 0.5M EDTA (pH 8.0)
Paraffin embedding

dependent variables

Cartilage matrix (stained with Alcian Blue)
Nuclei (stained with Fast Red)
Protein expression of ATF4, ATF3, CHOP, PPP1R15A, FGF21, and Sox9 (measured by immunofluorescence)

control variables

Not explicitly mentioned

controls

Positive controls: Not specified
Negative controls: Not specified

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