Anti rat igg alexa fluor 488

For microtubule observations, control and extract-treated root rips were excised and fixed in 4% (w/v) paraformaldehyde (PFA) solution in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO₄, pH 6.8) + 5% (v/v) dimethyl sulfoxide (DMSO) for 1 h. Fixed specimens underwent cell wall digestion with a 3% (w/v) Macerozyme R-10 + 3% (w/v) cellulase Onozuka R-10 (Duchefa Biochemie, Haarlem, The Netherlands) solution in PEM, for 90 min. After digestion, root tips were squashed gently on coverslips coated with poly-l-lysine and the released cells were left to dry and adhere. Afterwards, they were extracted with a 5% (v/v) DMSO + 1% (v/v) Triton X-100 solution in phosphate-buffered saline (PBS, pH 7.2), for 1 h. Rat anti-α-tubulin (YOL 1/34, Bio-Rad Laboratories, Hercules, CA, USA or Santa Cruz Biotechnology, Dallas, TX, USA) was used as primary antibody (diluted 1:50 in PBS, incubated overnight) and anti-rat IgG Alexa Fluor 488 (Cell Signaling Technology, Danvers, MA, USA) as secondary antibody (diluted 1:300 in PBS, incubated at 37 °C for 2 h). DNA counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, specimens were mounted with anti-fade medium (PBS 1:2 glycerol (v/v) + 0.5% (w/v) p-phenylenediamine).

This was performed as in ref. 17 (link), with modifications. M1 and M2 macrophages in aortic root sections were identified by co-staining for iNOS (#ab15323, Abcam) or mannose receptor (“CD206”, #64693, Abcam), respectively, and macrophage (CD68, #MCA1957GA, Bio-Rad), as follows: frozen sections were fixed in acetone (15 min, 4 °C), non-specific sites blocked, and this was followed by simultaneous incubation with antibodies against iNOS (1:100 dilution) and CD68 (1:300 dilution), or CD206 (1:100 dilution) and CD68 (1:300) for 3 hours. Sections were then incubated simultaneously with anti-rabbit IgG Alexa Fluor-555 (#4413 S, Cell Signaling) and anti-rat IgG Alexa Fluor-488 (#4416 S, Cell Signaling), both at 1:1,000 dilution (1 hour). Slides were mounted using the Prolong Gold Antifade Reagent with DAPI (Cell Signaling, #8961). All antibody dilutions were done using the IHC TEK antibody diluent (IW-1,000, IHC World).