Specimens were sectioned at a thickness of 3 μm and stained on positively charged glass slides stored at 4°C within 3 days after sectioning. Deparaffinization, rehydration, and antigen retrieval were performed by BERS2 (prediluted; pH 9.0) antigen retrieval solution (ref. AR 9640) performed on the Bond-III Leica automated slide stainer for 20 minutes at 100°C. Specimens were incubated with primary mouse anti–PD-L1 monoclonal antibody (ref. M365329; Dako) using a concentration of 1:10 for 2×60 minutes at room temperature, followed by visualisation with the Leica Bond detection kit for 20 minutes at room temperature (Ref. DS 9800). The specimens were then counterstained with haematoxylin and coverslipped. Each IHC run contained a positive control (tonsil tissue)
Bond detection kit
Manufactured by Leica
Sourced in China
The Leica Bond detection kit is a laboratory equipment product designed for the detection and identification of various biological molecules. It provides a reliable and efficient method for researchers to analyze and characterize their samples. The core function of this kit is to facilitate the detection and analysis of specific target molecules, enabling researchers to obtain accurate and reproducible results in their scientific investigations.
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6 protocols using bond detection kit
1
PD-L1 Immunohistochemistry Protocol
2
Immunohistochemical Profiling of Ovarian Tissue
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Serial ovarian tissue sections were purchased from Taibosi Bio (Xian, China). IHC staining was conducted using the Leica BOND-III platform (Leica, Wetzlar, Germany). Antigen retrieval was performed using BOND Epitope Retrieval Solution for 20 min at 100°C. Specimens were incubated with rabbit anti-UBE2B (1:200, 10733-1-AP), rabbit anti-RAD18 (1:200, DF7314, Affinity Biosciences, Changzhou, China), rabbit anti-ZMYM2 (1:200, GTX31821, GeneTex, Shenzhen, China) for 20 min at room temperature, followed by visualization with the Leica Bond detection kit at room temperature. The specimens were then counterstained with hematoxylin. Protein expression is scored following the methods used in the Human Protein Atlas (HPA) [19 (link)]. In brief, the IHC images were manually scored by two independent pathologists without authorships in this study. Staining intensity (negative, weak, moderate or strong) and the fraction of stained cells (<25%, 25–75% or >75%) were combined to give a final score as follows: negative (0), low (1), medium (2) and high (3).
3
Quantitative Immunohistochemistry for Cellular Signaling
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The phospho-S6 ribosomal protein (PS6; clone Ser235/236, Cell Signalling #2211) and phosphor-4E-BP1 (P4EBP-1; clone Thr37/46, 236B4, Cell Signalling #2855) antibodies were used in the dilution of 1:50 and 1:200, respectively. The HIER 30 ER2 antigen retrieval method was used and Leica Bond-Max automated immunohistochemistry for antigen detection was done as per the manufacturer’s protocol. Briefly, Leica bond detection kit (Leica #DS9800) for both antibodies was used containing the post primary (secondary) antibody—anti-mouse IgG (<10 μg/ml) in 10% (v/v) animal serum in tris-buffered saline/0.09% ProClin™ 950, DAB substrate chromogen and haematoxylin counterstain. A general control tissue micro-array was used for controls, which included a normal skin sample from excision of polydactyly in a newborn (presumed negative control). In addition, the controls for PS6 included a brain sample from FCD in a 5-year-old (positive control).
4
Histological Analysis of Pancreatic Islets
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Muscle sections were cut at 4 µm for histological analysis in hematoxylin and eosin (H&E) staining. Islets were identified using an anti-islet 1 antibody (clone EPR10362, ab178400, 1:250 dilution, Abcam) with human pancreas used as a positive control. Blocking was done with 1× Animal-Free Blocker (SP-5030-250, Thermo Fisher Scientific) in 2% NGS (5425S, Cell Signaling Technology) diluted in TBST (J77500.K2, Thermo Fisher Scientific). The primary antibody was diluted in blocking solution at a titer of 1:1,000. Heat-mediated antigen retrieval was done on the Leica BOND using EDTA-based pH 9 solution (AR9640, Leica). A Leica BOND detection kit (DS9800) was used (post-primary step was omitted) for DAB chromogenic staining.
5
Immunohistochemical Analysis of GALNT6
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The tissue microarray blocks were cut into 4-µm sections. Paraffin was removed with xylene, and antigens were retrieved using BOND epitope retrieval solution 1 (citrate buffer, pH 6.0; Leica) on a Leica BOND RX slide stainer for 30 min at 100 °C. Tissue sections were incubated with GALNT6-specific polyclonal antibodies (HPA011762, 1:150, Atlas Antibodies, Sigma) for 30 min, followed by visualization with the Leica Bond detection kit.
6
TMA-Based Immunohistochemical Analysis of SqCC
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We constructed a TMA of 90 cases of SqCC using 2‐mm cores. Next, 4‐μm sections of the TMA were cut and mounted on electrostatic slides for IHC analysis. The sections were heat‐dried at 56°C for 30 min, deparaffinized in xylene, and rehydrated with graded ethanol. All IHC assay procedures, including antigen retrieval and blocking of endogenous peroxidase activity, were performed automatically using the Bond‐III Leica automated slide stainer and primary antibodies, including Dako monoclonal mouse p63 (clone DAK‐p63) and TTF (both from Dako), Leica monoclonal mouse surfactant protein A (clone 32E12; Leica Biosystems), and Ventana monoclonal mouse p40 (clone BC28; Roche Diagnostics). A Bond‐III Leica microscope was used according to the manufacturer's protocol. The tissue sections were incubated with the primary antibody for 32 min at 42°C and then colorized using the Leica Bond detection kit. The specimens were counterstained with hematoxylin and then covered with a coverslip.
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