Alexa fluor 594 donkey anti rabbit igg h l

After deep anaesthesia, the superior pole of the eye was marked with a silk 6/0 suture to maintain proper orientation [1] (link), [3] –[5] (link), [73] (link), then rats were perfused through the heart with saline and 4% paraformaldehyde in 0.1 M PB, both eyes enucleated and further processed to obtain wholemounts or cross-sections, and the L- and S-cones were immunodetected by their specific opsin expression as described in detail [1] (link). In brief, retinal wholemounts or cross-sections were incubated in 1∶1000 goat anti-OPN1SW (N-20) (Santa Cruz Biotechnology, Heidelberg, Germany. Lot# L1906, sc-14363) and in 1∶1200 rabbit anti-opsin red/green (Chemicon-Milipore Iberica, Madrid, Spain. Lot # 2210352, AB 5405) and these were visualized with secondaries 1∶500 Alexa Fluor-488 donkey anti-goat IgG (H+L) (Molecular Probes-Invitrogen, Barcelona, Spain. Lot # 1182671, A11055), or 1∶500 Alexa Fluor-594 donkey anti-rabbit IgG (H+L) (Molecular Probes-Invitrogen, Barcelona, Spain. Lot # 1107500, A21207), respectively. All antibodies were diluted in phosphate buffered saline (PBS) containing 2% Triton X-100 (Sigma-Aldrich, Madrid, Spain).

Briefly, testis were embedded in paraffine and cut into 5-micrometer thick sections. After rehydratation, sections were processed as follows: Day 1: free-floating sections were rinsed in Tris-buffered saline (TBS; 0.25 M Tris and 0.5 M NaCl, pH = 7.5). Sections were incubated 30 min at 80 °C in 10 mM citrate buffer pH = 6 0,02% Tween, cooled down for 30 min and then rinsed 3 times in TBS. After 30 min incubation in 0.3% Triton X-100 in TBS, sections were rinsed 3 times in TBS and blocked in 3% BSA (or 3% donkey serum) in TBS for 1 hr. Slices were then incubated in 1% BSA, 0.15% Triton X-100 in TBS for 12 to 72 hours at 4 °C with primary antibodies against TRA98 (1:1000 kindly provided by B. Boizet) or HA (1:500, 715500, Invitrogen). Day 2: sections were rinsed 3 times for 10 min in TBS and incubated for one hour with secondary antibodies Alexa Fluor® 594 Donkey Anti-Rabbit IgG (H+L) (1:1000, A-21207 Molecular Probes) and Alexa Fluor® 488 Donkey Anti-Rat IgG (H+L) (1:1000, A-21208 Molecular Probes). Nuclei were colored by DAPI staining (1:5000). Sections were rinsed for 10 min twice in TBS and twice in 0.25 M Tris buffer (0.25 M Tris, pH = 7.5) before mounting slides with FluorSave^TM Reagent (345789, Merck Millipore).
Confocal sections were acquired using confocal microscopy (LSM780, or AxioImager Z1-Dr, Zeiss).

Anti-TRIM44 polyclonal antibody (Proteintech Group, 11511–1-AP); anti-Ub antibody (Biolegend, 646301); anti-mCherry antibody (ThermoFisher, PA5–34974); anti-NFE2L2 antibody (ThermoFisher, PA5–27882); anti-ATG5 antibody (Novus Biologicals, NBP2–24389); anti-GFP antibody (Santa Cruz Biotechnology, sc-9996); anti-ACTB antibody (Santa Cruz Biotechnology, sc-47778), anti-HA antibody (Santa Cruz Biotechnology, sc-805); anti-LC3B antibody (Cell Signaling Technology, 3868); anti-SQSTM1 antibody (Cell Signaling Technology, 88588); anti-LaminB1 antibody (R &D, MAB8525); anti-KEAP1 antibody (Origene, TA502059); anti-GAPDH antibody (Invitrogen, PA5–85074); anti-FLAG antibody (Invitrogen, PA1–984); anti-mTOR antibody (CST, 2972); anti-phospho-mTOR (Ser2448) antibody (Millipore Sigma, 09–213) anti-SQSTM1 antibody(CST, 7695,88588); anti-phospho-SQSTM1/p62 (Ser349) (E7M1A) antibody (CST, 16177S); anti-phospho-SQSTM1/p62 (Ser403) (D8D6T) antibody (CST, 39786S); anti-PKA Cα antibody (CST, 4782); ; anti-PKA substrate antibody (CST, 9624); Goat anti-Mouse IgG (Invitrogen, 31430); Goat anti-Rabbit IgG (Invitrogen, 31460); Alexa Fluor 594 donkey anti rabbit IgG (H + L) (Invitrogen, A21207); Alexa Fluor 546 goat anti mouse IgG (H + L) (Invitrogen, A11003).

Cells were fixed via 4% paraformaldehyde (Sigma) for 15 minutes at room temperature. After washed with PBS, cells were permeabilized by 0.5% Triton X‐100 (Sigma) for 15 minutes at room temperature. Cells were washed and incubated with 4% bovine serum albumin (BSA) for 1 hour at room temperature, which were then stained with primary antibodies at 4℃ overnight. These primary antibodies were used: OCT4, SOX2, SSEA4, TRA‐1‐81, AFP, SMA, NESTIN (1:200; Abcam). We washed the cells and incubated them with the secondary antibodies for 1 hour at room temperature, which were as following: Alexa Fluor 488 Goat anti‐Mouse IgG (H + L; 1:500; Invitrogen) and Alexa Fluor 594 Donkey anti‐Rabbit IgG (H + L; 1:500; Invitrogen). The cells were washed and Nuclei were stained with DAPI (1 μg/mL; Life Technology) for 10 minutes. The stained cells were observed through a confocal microscope (Nikon). Alkaline phosphatase (AP) staining was performed by Alkaline Phosphatase Assay Kit (Beyotime) according to the manufacturer's instruction, and cells were analysed via a microscope (Leica).