Alexa fluor 568 goat anti rabbit immunoglobulin g igg

Samples, including cell culture samples and cryosectioned tissues, were rinsed with 1 × PBS (Gibco) and treated with 0.3% Triton X-100 (Sigma-Aldrich) to increase permeability. After incubation in blocking buffer comprised of 1 × PBS (Gibco), 1% BSA (Sigma-Aldrich), and 0.3% Triton X-100 (Sigma-Aldrich) for 1 hour, samples were incubated with the diluted primary antibodies (1:100) followed by the corresponding secondary antibodies (1:200). Cell nuclei were stained with DAPI (Santa Cruz). Samples were mounted using an anti-fluorescence quenching agent (Solarbio, Beijing, China) and imaged by confocal microscopy (Nikon).
The primary antibodies include rabbit polyclonal anti-RFP (Abcam, ab185921), rabbit polyclonal anti-Cx43 (Abcam, ab11370), rabbit polyclonal anti-TIMP3 (Abcam, ab39184), rabbit polyclonal anti-SOX2 (Abcam, ab97959), rabbit monoclonal anti-Nanog (Abcam, ab109250), and rabbit polyclonal anti-LAMA4 (Abcam, ab209675). The secondary antibody was Alexa Fluor 568 goat anti-rabbit immunoglobulin G (IgG; Invitrogen).

Samples including fixed cells and cryosectioned tissues were rinsed with 1×PBS (Gibco), treated with 0.5% Triton X-100 (Thermo), and incubated in Superblock (Thermo) for 1 hour. Samples were incubated with the diluted primary antibodies (1:200) at 4°C overnight followed by the incubation of the corresponding secondary antibodies (1:200) at room temperature for 1 hour. Samples were mounted using ProLong Gold Antifade reagent with 4’,6-diamino-2-phenylindole (DAPI; Invitrogen) and imaged by confocal microscopy (40×; Nikon; Laser power: 15.0–20.0; PMT HV: 110–138; Acquisition times: within 6 minutes).
The primary antibodies include rabbit polyclonal anti-matrix metalloproteinase-3 (MMP3), rabbit polyclonal anti-tissue inhibitor of metalloproteinases (TIMP3), and rabbit polyclonal/mouse monoclonal anti-myocilin (MYOC). Details were listed in Table 1. The secondary antibody was Alexa Fluor^® 568 goat anti-rabbit immunoglobulin G (IgG; Invitrogen).Table 1

Antibodies for IHC and WB

Name	Brand	Catalog No.	Application	Dilution
TIMP3 (Rabbit polyclonal)	Abcam	ab39184	IHC	1:200
MMP3 (Rabbit polyclonal)	Abcam	ab53015	IHC	1:200
MYOC (Rabbit polyclonal)	Abcam	ab41552	IHC	1:200
MYOC (Mouse monoclonal)	Abnova	H00004653-M01	WB	1:1000
GAPDH (Rabbit monoclonal)	Abcam	ab181602	WB	1:5000