Goat anti rabbit igg

The immunofluorescence (IF) technique was used to detect the M1- and M2-phenotype TAMs in tumor microenvironment. Rabbit anti-mouse iNOS (Bioss, Woburn, MA, USA) and goat anti-mouse arginase 1 (Santa Cruz, Dallas, TX, USA) were used as primary antibodies in order to label M1 and M2 macrophages, respectively. The secondary antibodies included goat anti-rabbit IgG (Bioss, Woburn, MA, USA) and donkey anti-goat IgG (Santa Cruz, Dallas, TX, USA). In the IF examination, tumor specimens were harvested, fixed in 4% formalin for 48 h, embedded in paraffin, sectioned, deparaffinized in xylene, and rehydrated in ethanol. After antigen retrieval by boiling in 10 mM Tri-EDTA (pH 8.0) for 1 h, the sections were washed with PBST (1 × phosphate-buffered saline (PBS), 0.1% Tween 20), incubated with primary antibodies, washed again with PBST, treated with secondary antibodies, and left in the dark site for 1 h. The slides were observed and pictured by an upright fluorescence microscopy (Olympus BX51, Olympus, Tokyo, Japan). The pictured images were analyzed and merged by ImageJ software (NIH, Bethesda, MD, USA). ImageJ with the colocalization and color deconvolution plugins were also used to quantify immunofluorescence and chromogenic signal intensity on image.

Primers for RPA were designed using NCBI Primer-BLAST¹, and in vitro transcribed (IVT) templates for crRNA were ordered from Genewiz (Suzhou, China). Commercial Cas9, Cas12a, Cas13a, and FAM-TTTTT-Quencher used in fluorescent reporter assay and FITC-TTTTTT-Biotin probe used in gold nanoparticle-labeled lateral flow strips were obtained from Biolifesci (Guangzhou, China). The detailed sequences are listed in Supplementary Table 2. M. hominis genomic DNA was extracted using lysis buffer for Microorganism to Direct PCR (Takara, Tokyo, Japan). The TwistAmp^® Basic kit for RPA test was purchased from TwistDx (Cambridge, United Kingdom). T7 RNA polymerase, the NTP mix, and the RNA purification kit were purchased from New England Biolabs (Ipswich, MA, United Kingdom). RNase-free water and Recombinant DNase I (RNase-free) were purchased from Takara. RNase-free water was used in all experiments. Streptavidin was purchased from Solarbio (Beijing, China), while Goat anti-Rabbit IgG and gold-nanoparticles–Rabbit anti-FITC were purchased from Bioss (Beijing, China). The fluorescence quantifications were measured with a Wallac 1420 plate reader (PerkinElmer, United States). Amplification was confirmed by 3% agarose gel electrophoresis and the RPA products were visualized using a Gel Doc system (Bio-Rad, United States). The relevant reagents are presented in Supplementary Table 1.

We used 1% Triton lysis buffer to dissolve the cells. Finally, the samples were separated by preprepared SDS-PAGE electrophoresis after adding protein loading buffer, and then, the gel was transferred to PVDF membrane for Western blotting analysis. PGSK3β (sc-81495) was purchased from Santa Cruz Biotechnology, USA. β-Catenin (bs: 1165R), Cyclin D1 (bs: 20596R), and Goat Anti-rabbit IgG (bs: 0295G) were purchased from Bioss (China, Beijing). MiniChemi™ 500 small chemiluminescence imaging and analysis device were used to process Western blotting images. (Sage Creation Science, Beijing, China).

Protein was extracted by RIPA Lysis buffer (Sangon, Shanghai, China), subjected to SDS-PAGE gel, and transferred to PVDF membranes. The membranes were incubated with anti-p-glycoprotein (anti-p-gp, 1:500, Bioss, Beijing, China), anti-myeloid cell leukemia-1 (anti-MCL-1, 1:1,000, Bioss), anti-FBN1 (1:1,000, Bioss), anti-β-catenin (1:2,000, Bioss), anti-c-Myc (1:1,000, Bioss) or anti-β-actin (1:2,000, Bioss). After incubating with Goat Anti-Rabbit IgG (1:20,000, Bioss), the signals were determined by Enhanced ECL Chemiluminescence Detection Kit (Vazyme, Nanjing, China).

We performed protein extraction and Western blotting analyses as previously described [32 (link)]. In the test, we used antibodies including monoclonal rabbit-anti-rat Ras (1:1000, Bioss Biotechnology, Beijing, China), rabbit-anti-rat p65 (1:1000, Bioss Biotechnology), rabbit-anti-rat p-IκB (1:500, Bioss Biotechnology, Beijing, China), and rabbit-anti-rat β-actin (1:10000, Bioss Biotechnology, Beijing, China). Goat-anti-rabbit IgG (Bioss Biotechnology, Beijing, China) was used as a secondary antibody. Gray-scale images and quantification were analyzed with Image-J software (National Institutes of Health, Bethesda, MD, USA).