Goat anti mouse igm alexa488

Cells were grown in 12-well plates (Greiner Bio-One) on coverslips (Ø 20 mm; Thermo Scientific) to 60–80% confluence. Cells were washed twice with PBS and fixed with 4% formaldehyde in PBS for 10 min at room temperature. Fixed cells were stored at 4 °C in PBS-NaN₃ (0.03%) or washed three times with PBS+/+ (Gibco) and directly used for immunocytochemistry. Subsequently, cells were permeabilized (0.1% Triton X-100 and 0.2% BSA in PBS) for 15 min and blocked (2% BSA-PBS) for 30 min at room temperature. Primary and secondary antibodies (2% BSA-PBS) were incubated for 1 h at room temperature. coverslips were washed and mounted onto glass slides using DABCO-glycerol medium (Sigma-Aldrich) containing DAPI (1:10,000; Sigma-Aldrich) in order to counterstain nuclei. The anti-glycogen antibody (1:500) was courtesy of Dr. O. Baba (Tokyo Medical and Dental University, Tokyo, Japan), and the secondary antibody was goat anti-mouse IgM Alexa647 (1:200; Invitrogen) or goat anti-mouse IgM Alexa488 (1:200; Invitrogen).

Immunofluorescence staining was carried out according to standard protocols. Primary antibodies used were anti-OCT4 (#sc5279 Santa-Cruz, Heidelberg, Germany, 1:200), anti-NANOG (#sc293121 Santa-Cruz, 1:200), anti-TRA1-60 (MAB4360 Millipore, Darmstadt, Germany, 1:250) and anti-SSEA-4 (MAB4304 Millipore; 1:250). Secondary antibodies used were goat anti-mouse IgG Alexa-546 (#A-11030 Invitrogen, Schwerte, Germany) and goat anti-mouse IgM Alexa-488 (#A-21042 Invitrogen). Nuclei were co-stained with 4,6-diamidino-2-phenylindole DAPI (1:2000). Alkaline Phosphatase staining was performed using the Alkaline Phosphatase Staining Kit II (#00-0055 Stemgent, Glasgow, United Kingdom) according to the manufacturer’s instructions.

Glomerular heparanase and HS expression was determined by indirect immunofluorescence staining on cryosections (2 μm) as described [22 (link)]. Primary antibodies included heparanase (HPA1, ProsPecTany, Rehovot, Israel) and a mouse monoclonal anti-HS antibody, JM403 [23 (link)]. Secondary antibodies included goat anti-rabbit IgG Alexa 488 and goat anti-mouse IgM Alexa 488 (Invitrogen Life Technologies, Breda, The Netherlands). Glomerular heparanase and HS staining intensities were scored in 50 glomeruli per section on a scale between 0 and 10 (0 = no staining, 5 = 50% staining, 10 = 100% staining). Scoring was performed by two independent investigators on blinded sections using a Leica CTR6000 microscope.