Donkey anti rabbit igg h l alexa fluor 488

HA-Tag Rabbit mAb; CST; 3724; Clone C29F4; Lot 8; IF/WB; 1:1500/1:1000
Calnexin Antibody (H-70); SCBT; sc-11397; WB; 1:10,000
Mouse anti-α-Tubulin antibody; Sigma-Aldrich; T6074; Clone B-5-1-2; WB; 1:10,000
Donkey Anti-Rabbit IgG H&L Alexa Fluor^® 488; Abcam; ab150073; IF; 1:1000
Anti-rabbit IgG, HRP-linked Antibody; CST; 7074; Lot 28; WB; 1:6000
Anti-mouse IgG, HRP-linked Antibody; CST; 7076; Lot 33; Dot blot; 1:4000
Phalloidin-Tetramethylrhodamine B isothiocyanate; Sigma Aldrich; P1951; IF; 50 μg/mL.

The 5th-passage MDSCs, FFCs, and EFCs (1 × 10⁴ cells/well) were cultured onto coverslips and fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeabilized with 0.1% Triton X-100 for 15 min on ice. Then, cells were blocked in 5% bovine serum album for 1h at 37 °C and incubated with primary antibodies overnight at 4 °C. This was followed by incubation with secondary antibodies (Donkey Anti-Rabbit IgG H+L (Alexa Fluor^® 488) (Abcam, Carlsbad, CA, USA) for 1h in the dark. The cells were incubated with DAPI for 20 min to stain nuclei, and cell immunofluorescence images were acquired using a Nikon microscope (Nikon-Air, Nikon instruments Co., LTD, Tokyo, Japan). Negative controls were incubated with PBS instead of the primary antibody. See Supplementary Table S1 for a list of the primary antibodies used.

661w cells were grown on confocal plates and fixed and permeabilized with methanol for 15 min at −20 °C, followed by three washes with PBS. Then the cells were incubated for 1 h in blocking buffer (5% normal goat or donkey serum, 0.3% Triton X-100 in PBS), following by incubation with anti-LC3B primary antibody (1:400, Cell Signaling Technology, MA, USA, Cat#83506), anti-TOM20 primary antibody (1:200, Cell Signaling Technology, Cat#42406), anti-PINK1 primary antibody (1:200, Abcam, Cambridge, UK, Cat#ab23707) and anti-Parkin primary antibody (1:200, Abcam, Cat#ab77924) overnight at 4 °C. After gently washing, the respective fluorescent secondary antibodies purchased from Abcam (Goat Anti-Mouse IgG H&L, Alexa Fluor 488, Cat#ab150113, 1:1000; Goat Anti-Rabbit IgG H&L, Alexa Fluor 647, Cat#ab150079, 1:1000; Donkey Anti-Mouse IgG H&L, Alexa Fluor 594, Cat#ab150108, 1:1000; Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488, Cat#ab150073, 1:1000) were added and the cells were incubated at 37 °C for 2 h in the dark. Nuclei were stained with 0.01 mg/ml Hoechst 33342. The cells were photographed using a laser-scanning confocal microscope (Leica, Nussloch, Germany) for fluorescence and a digital camera driven by LAS AF software.

After treatment, HUVECs were fixed with 4% paraformaldehyde. Cells were then incubated with phospho‐Histone H3‐S10 antibody (Cat#: AP0840, ABclonal Technology, Wuhan, China) overnight at 4°C. Cells were then washed with phosphate‐buffered saline (PBS) three times. Then, cells were incubated with the secondary antibodies Donkey Anti‐Rabbit IgG H&L (Alexa Fluor® 488) (Cat#: ab150065, Abcam, USA) or Goat anti‐rabbit IgG (H + L) Highly Cross‐Adsorbed Secondary Antibody, Alexa Fluor™ 546 (Cat#: A‐11035, Invitrogen, USA). After being washed three times, cells were mounted with an antifading Mounting Medium with DAPI (Cat#: S2110, Solarbio, Beijing, China). Phospho‐Histone H3‐positive cells were captured with a Leica TCS SP5 II confocal microscope (Leica, Germany), and Phospho‐Histone H3‐positive cells were counted by Image J (NIH, USA).

Double immunofluorescent staining was performed using the following primary antibodies: mouse anti-NF-κB p65 (dilution ratio: 1:150; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CTSK (1:150; Abcam), rabbit anti-RANKL (1:150; Proteintech Group Inc., Rosemont, IL, USA), rabbit anti-OPG (1:150; Abcam), rabbit anti-OPN (1:200; Abcam), and rabbit anti-OCN (1:200; Abcam). The sections were first blocked using 5% bovine serum albumin and were then incubated with a specific primary antibody, followed by species-matched secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor 594 and Donkey Anti-Rabbit IgG H&L Alexa Fluor 488; Abcam), at a 1:200 dilution. All tissue sections were subjected to autofluorescence quenching (Vector TrueVIEW; Vector Laboratories, Inc.) and mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) following the manufacturer’s protocol. Fluorescent images were acquired using a Leica TCS-SP8 confocal laser scanning microscope (Leica Biosystems, Wetzlar, Germany) within 48 h after mounting. The immunofluorescence expression of each sample was evaluated using the MFI; a.u.).

Human cells were resuspended in a staining buffer (PBS + 0.1% bovine serum albumin +0.1% sodium azide) and treated with human FcR blocking agent (Miltenyi Biotec, cat. no. 130–059-901) for 10 min at 4°C prior to staining. Samples were stained with primary Anti-beta2-adrenergic receptor Ab (Abcam, cat. no. ab61778) for 30 min at 4°C. After three washing steps with a staining buffer, Donkey anti-rabbit IgG H + L (AlexaFluor488) (Abcam, cat. no. ab150073) was added, together with the viability stain 7-AAD (BioLegend, cat. no. 420404) and the remaining conjugated antibodies [CD19 PE (BioLegend, cat. no. 363004), CD14 PE-Vio770 (Miltenyi Biotec, cat. no. 130–098-074) and CD3 APC (BioLegend, cat. no. 3004399)] for 20 min 4°C protected from light. Samples were washed three times prior to analysis on a flow cytometer. Along with the samples, appropriate controls were prepared, including unstained control and compensation controls using BD™ CompBeads Anti-Mouse Ig, κ/Negative Control (BD Bioscience, cat. no. 552843). Secondary only controls were prepared by omitting the primary Anti-beta2-adrenergic receptor Ab staining step. The analysis of the stained samples was done using a BD FACSCantoTM II flow cytometer and computer equipped with the BD FACSDivaTM software (BD Biosciences, version 6.1.3). Data was analysed by the FlowJo software (version 10.6.1).