Immunostaining of Mouse Sinoatrial Node Tissues

Heart samples harvested from mouse SAN tissues were embedded in a tissue freezing medium, followed by sectioning at 10 μm using a cryostat (Leica CM3050 S). The slides were subjected to immunostaining using a previously described protocol Sun et al., 2009 (link). The following antibodies were used: anti-HCN4 (Millipore, Cat#: AB5805; Novus biologicals, Cat#: NB100-74439) at 1:200, anti-DNAJB6 (Novus, Cat#: H00010049-M0; Santa Cruz Biotechnology Inc, Cat#: sc-104204) at 1:200, anti-TBX3 (abcam, Cat#: ab99302). All images were captured either using a Zeiss Axioplan II microscope equipped with ApoTome and AxioVision software (Carl Zeiss Microscopy) or a Zeiss LSM 780 confocal microscope. Signal intensity from DNAJB6 and TBX3 antibodies immunostaining was quantified using Zen 2.3 Pro software.

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Ding Y., Lang D., Yan J., Bu H., Li H., Jiao K., Yang J., Ni H., Morotti S., Le T., Clark K.J., Port J., Ekker S.C., Cao H., Zhang Y., Wang J., Grandi E., Li Z., Shi Y., Li Y., Glukhov A.V, & Xu X. (2022). A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene. eLife, 11, e77327.

Publication 2022

CM3050 S anti-HCN4 anti-DNAJB6 anti-TBX3 Axioplan II microscope ApoTome AxioVision LSM 780 confocal microscope

Antibodies Biologicals Confocal microscope Dnajb6 Heart Microscopy Mouse Novus Tissue

Corresponding Organization : WinnMed

Other organizations : Qingdao University, Affiliated Hospital of Qingdao University, University of Wisconsin–Madison, University of California, San Francisco, XinHua Hospital, Xiangya Hospital Central South University, Central South University, Jiading District Central Hospital, Shanghai University, University of California, Davis, University of California, Irvine, University of Maryland, Baltimore, The University of Texas Health Science Center at Houston

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Variable analysis

independent variables

Antibody used for immunostaining (anti-HCN4, anti-DNAJB6, anti-TBX3)

dependent variables

Signal intensity from DNAJB6 and TBX3 antibodies immunostaining

control variables

Mouse SAN tissue samples
Tissue freezing medium
Cryostat sectioning at 10 μm
Immunostaining protocol (as described in Sun et al., 2009)
Microscope and imaging software (Zeiss Axioplan II with ApoTome and AxioVision or Zeiss LSM 780 confocal microscope)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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