3 3 di aminobenzidine

Mouse tissues were fixed with 10% formalin, dehydrated, and embedded into paraffin blocks. The paraffin-embedded specimens were sectioned at a thickness of 4 μm using a microtome (Leica, RM2235, United States). The sections were dewaxed and stained with H&E. After being dewaxed and antigen repaired, the sections were stained for IHC and IF staining. The primary antibodies used in IHC were Rabbit-anti Ki67 (1:300; Abcam, Cambridge, United States) and Rabbit-anti FTL (1:100; Proteintech, China). The secondary antibodies and 3, 3′-diaminobenzidine were purchased from Boster Bio (Pleasanton, CA, United States). In IF staining, Rabbit-LC3 (1:250, Proteintech) was used as the primary antibody, and Goat-anti rabbit IgG H&L (Alexa Fluor 488, 1:400; Abcam) was used as the secondary antibody.

Proteins were characterized by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The amounts of proteins were determined following the Bradford method with bovine serum albumin as standard [43 ]. Protein gels were either stained with Coomassie brilliant blue R-250 or used for Western blot analysis with an anti-MtNFH1 antibody [38 (link)]. Goat–anti-rabbit IgG antiserum coupled to horseradish peroxidase was used as second antibody, and blots were developed with 3,3′-diamino-benzidine (Boster, Wuhan, China).

The tissue samples were embedded in paraffin, cut into 5‐μm sections, dewaxed in xylene, and rehydrated in alcohol. After treatment with citrate buffer for antigen retrieval and treatment with 1% bovine serum albumin for 1 h, the sections were hybridized with anti‐CCND1 (1:200; Abcam Cat: ab16663; RRID: AB_443423; Abcam Inc., Cambridge, MA, USA) at 4℃ overnight and then incubated with horseradish peroxidase (HRP)‐labeled immunoglobulin G (IgG, 1:1,000; Abcam Cat#: ab6721; RRID: AB_955447; Abcam Inc) at 25℃ for 1 h. The staining was developed by 3,3′‐diaminobenzidine (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The nuclei were stained by hematoxylin (Servicebio, Wuhan, Hubei, China). After that, the section slides were sealed and observed under a microscope.

Liver cancer TMAs composed of 110 FFPE tissue Sects. (4 μm thick) were prepared the same way as previously described by Guttà C et al. [31 (link)] TMA IHC staining was conducted based on a standard approach. Briefly, the sections were deparaffinized using xylene, rehydrated with an ethanol gradient, treated to quench peroxidase activity, blocked with 5% normal goat serum, and incubated with anti-YAP/GLUT1 (1:200) overnight at 4 °C. As a negative control, the antibody was omitted. The slides were then probed with biotin-labeled goat anti-rabbit IgG, treated with a streptavidin peroxidase solution (SABC kit, Boster, Wuhan, China), and then 3,3-diaminobenzidine (Boster, Wuhan, China) in PBS with 0.05% H2O2 was used to treat the samples for 5 min at the room temperature for color development, after which the samples were counterstained using hematoxylin.

Proteins were separated by SDS-PAGE on 12% polyacrylamide gels and stained with Coomassie Brilliant Blue G-250. For Western blot analysis, proteins were separated onto nitrocellulose membranes. Membranes were incubated with commercially available antibodies against protein tags, with an antibody recognizing NopL of Sinorhizobium sp. NGR234 (Zhang et al., 2011 (link)) or against an antibody recognizing a C-terminal part of NopD of strain Bradyrhizobium sp. XS1150. For preparation of the anti-NopD antibody, recombinant NopD (residues 640–1017) with an N-terminal 6 × His tag was expressed in E. coli BL21 (DE3) and the purified protein was used for immunization of a rabbit. After incubation with horseradish peroxidase-conjugated second antibodies, Western blots were developed with 3,3′-diaminobenzidine (Boster, Wuhan, China) or by electrochemiluminescence detection reagents (Amersham GE Healthcare, Little Chalfont, United Kingdom) according to the supplier’s protocols.

An ESCC TMA, containing a total of 118 formalin-fixed paraffin-embedded tissue samples, was constructed according to a previously described method (10 (link)). IHC was also performed according to a previously described method (11 (link)). Briefly, the sections were deparaffinized in xylene and rehydrated through a gradient concentration of alcohol. The endogenous peroxidase activity was inactivated, non-specific staining was blocked by 5% normal goat serum and all sections were incubated with anti-p53 antibody (1:100; Abmart Inc.) overnight at 4°C. The slides were incubated with biotin-labeled goat anti-rabbit immunoglobulin G and further incubated with streptavidin peroxidase solution (SABC kit, Boster Biological Technology, Ltd., Wuhan, China). The staining was visualized by reaction with 3, 3′-di-aminobenzidine (Boster Biological Technology, Ltd.) in phosphate-buffered saline [PBS; Dycent Biotech (Shanghai) Co. Ltd., Shanghai, China] with 0.05% H₂O₂ for 5 min at room temperature. Control staining was performed by staining the same TMA (duplicate) with PBS rather than anti-p53 and no immunostaining was observed. The slides were counter-stained with hematoxylin, washed in double-distilled H₂O and mounted with resinous mounting medium. The TMA were scored separately by two pathologists who had no prior knowledge of the clinicopathological status of the specimens on the TMA.