Alexa fluor 594 conjugated goat anti mouse

TNF-α- and IL-1β-treated rat chondrocytes were rinsed in PBS and then fixed with 10% neutral-buffered formalin for 30 min at 21 °C. Cells were incubated with Triton X-100 (Beyotime Biotechnology, Beijing, China) for 10 min to penetrate the cell membrane and donkey serum (Beyotime Biotechnology) for 1 h to block nonspecific binding sites. Cultured cells were incubated with primary antibodies against type II collagen (ab34712; Abcam; 1:100), SOX9 (ab185966; Abcam; 1:100), and LRP3 (sc-373736; Santa Cruz Biotechnology, USA; 1:50) at 4 °C overnight. The cells were then washed with PBS three times and incubated with Alexa Fluor 488-conjugated goat anti-rabbit (A-11008, Thermo Fisher Scientific; 1:200) or Alexa Fluor 594-conjugated goat anti-mouse (A-11005, Thermo Fisher Scientific; 1:200) secondary antibodies for 1 h at 21 °C. Nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China) for 10 min. Finally, the samples were rinsed with PBS and visualized using a confocal microscope (Olympus Life Science, Tokyo, Japan).

We cultured PAEC cells in μ-Slide 8-well chambers (ibidi, Gräfelfing, Germany) and performed the KD experiments by adjusting the volumes. After KD, cells were washed three times in PBS before being fixed with 4% formalin for 10 min at 37°C. After washing the cells six times in PBS, we proceeded to permeabilize them in PBS + BSA 1% (w/v) containing 0.1% Triton X (v/v). Then, we blocked them in PBS + BSA 2% (blocking buffer) for 1 h at room temperature (RT). We incubated the cells with the primary antibodies overnight in blocking buffer, washed three times with blocking buffer for 5 min, and incubated with the secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) in blocking buffer for 1 h in the dark. Finally, we washed the chambers three times for 5 min in PBS and mounted them in ProLong Diamond Antifade Mountant (ThermoFisher, Waltham, USA). Images were acquired using a Leica DMI6000 inverted microscope with an integrated confocal module SP5 (Leica Microsystems, Germany). The settings used for confocal imaging were maintained in the samples
The following antibodies and dilutions were used are as follows: anti-ET-1 (Abcam, Cambridge, UK, #ab2786, 1:500), phalloidin-Alexa488 (Abcam, #ab22744, 1:1,000), and Alexa Fluor 594-conjugated goat anti-mouse (ThermoFisher, Waltham, USA, #A-11005, 1:1,000).

Immunofluorescence was performed following [29 (link)]. Briefly, the cells were fixed in 4% formaldehyde (PFA) for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 (#194854; MP Biomedicals, USA) for 10 min and 0.1% saponin (#S7900; Sigma Aldrich, Czech Republic) for 12 min. After that, the dishes were washed twice in phosphate-buffered saline for 15 min. We used 1% bovine serum albumin (BSA; #A2153-506; Sigma Aldrich, Czech Republic) dissolved in 1× PBS as a blocking solution. Next, the samples were incubated in blocking solution for one hour at room temperature and then washed in 1× PBS for 15 min. For immunofluorescence analysis, the following antibodies were used: anti-ACE2 (#ab15348; Abcam, UK) and α-actinin (#A7811; Sigma Aldrich, Czech Republic). As secondary antibodies, we used the following: Alexa Fluor 594-conjugated goat anti-rabbit (#A11037; ThermoFisher Scientific, Czech Republic), Alexa Fluor 594-conjugated goat anti-mouse (#A11032; ThermoFisher Scientific, Czech Republic), Alexa Fluor 488-conjugated goat anti-rabbit (#ab150077; Abcam UK) antibodies. The negative control was considered samples incubated without primary antibodies. Cell nuclei (GC-rich sequences of DNA) were stained with 4′,6-diamidino-2-phenylindole (DAPI; Merck, Czech Republic). As a mounting medium, we used Vectashield (#H-1000, Vector Laboratories, USA).