IHC staining on the respective formalin-fixed, paraffin-embedded tissue sections was performed using the Leica BOND-MAX and Ventana Benchmark XT autostainers according to the conditions stated in Table 1 . Tissue sections of 5 µM underwent automated deparaffinization followed by incubation with their optimized antigen retrieval solutions. Slides were then incubated with antibody as indicated in Table 1 . Detection of antibody staining was performed according to manufacturer’s protocol for the detection kits used with an extension of hematoxylin counterstain extended to 10 min to ensure for a defined stain. Slides were rinsed with deionized water followed by manual mounting of coverslips. Positive and negative controls were included in each run, consisting of tissue with known expression and tissue stained without primary antibody, respectively. Quantification was assessed double blind by a trained pathologist (B. Pang) and expressed as an H-score. Melanoma samples were purchased from BioMAX-ME2082A.
Bond max
Manufactured by Roche
The Bond-Max is a laboratory instrument designed for automated DNA and RNA extraction and purification. It utilizes magnetic bead-based technology to isolate and purify nucleic acids from a variety of sample types. The Bond-Max system is capable of processing multiple samples simultaneously to increase efficiency and throughput.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark xt, by Roche (3 mentions)
Synaptophysin, by Beijing Strong Biotechnologies (2 mentions)
Napsina, by Beijing Strong Biotechnologies (2 mentions)
Ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Ventana autostainer, by Roche (1 mentions)
Pd l1 clone 22c3, by Merck Group (1 mentions)
Kaluza v2, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Ab5603, by Merck Group (1 mentions)
Hpa001906, by Merck Group (1 mentions)
Ventana discovery, by Roche (1 mentions)
Dia h09, by Dianova (1 mentions)
Pd l1 clone 22c3, by Agilent Technologies (1 mentions)
8 protocols using bond max
1
Immunohistochemical Staining Protocol
2
Immunohistochemical Quantification of Protein Expression
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Immunohistochemistry (IHC) staining on the respective formalin‐fixed, paraffin‐embedded tissue sections was performed using the Leica BOND‐MAX and Ventana Benchmark XT autostainers according to the conditions stated in Table 1 . Tissue sections underwent automated deparaffinization followed by incubation with their optimized antigen retrieval solutions. Slides were then incubated with antibody as indicated in Table 1 . Detection of antibody staining was carried out according to the manufacturer's protocol for the detection kits used with an extension of hematoxylin counterstain extended to 10 min to ensure for a defined stain. Slides were rinsed with deionized water followed by manual mounting of coverslips. Positive and negative controls were included in each run, consisting of tissue with known expression and tissue stained without primary antibody, respectively. Quantification was assessed double‐blind by a trained pathologist (BP) and expressed as an H‐score. The H‐score was determined by the formula 3 × percentage of strongly staining cells, 2 × percentage of moderately staining cells, and 1 × percentage of weak staining cells, giving a range of 0–300 for the H‐score.
3
Differentiating Pulmonary Enteric Adenocarcinoma from Metastatic Colorectal Cancer
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To evaluate if the pulmonary enteric adenocarcinoma samples could also be differentiated from metastatic colorectal cancer ("primary pulmonary" vs. "metastatic") by means of conventional histomorphology and immunohistochemistry, histology was reevaluated by five senior pathologists blinded to clinical information.
Immunohistochemical staining for ALK, CDX2, CK7, CK20, and TTF-1 was performed on the Leica BOND-MAX and Ventana BenchMark XT automated slide stainer according to the manufacturer's instructions. Antibodies and their according manufacturers as well as concentrations are shown in Supplementary Table S2.
Immunohistochemical staining for ALK, CDX2, CK7, CK20, and TTF-1 was performed on the Leica BOND-MAX and Ventana BenchMark XT automated slide stainer according to the manufacturer's instructions. Antibodies and their according manufacturers as well as concentrations are shown in Supplementary Table S2.
4
Comprehensive Immunophenotyping of FFPE Samples
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4 μm-thick sections extracted from representative FFPE blocks of each case were evaluated for a series of immunohistochemical staining, including cytokeratin 7 (CK7, clone RN7, BIO), TTF-1 (clone 8G7G3/1, ZECA), NapsinA (polyclonal, MXB), P63 (clone UMAB4, BIO), P40 (clone ZR8, MXB), cytokeratin 5/6 (CK5/6, clone D5/16B4, MXB), CD56 (clone UMAB83, BIO), synaptophysin (polyclonal, MXB), chromogranin A (clone EP38, BIO), Pan-CK (clone AE1/AE3, BIO), P53 (clone DO-7, BIO), Rb (clone 13A10, CELNOVTE), Ki-67 (clone MIB-1, DAKO) and PD-L1 (clone 22C3, MERCK). IHC staining was performed by the 2-step Envision procedure using Leica Bond-Max or Roche Ventana autostainer. Related antibodies, together with their clone, dilutions and machine platform was listed on supplementary Table 1 . The positive percentage and expression pattern (membrane, cytoplasm or nucleus) were evaluated. Immunophenotype of a tumour was regarded as positive when it expressed moderate or greater intensity in more than 10% of neoplastic cells and reactivity less than 10% cells were considered as negative (excepted for PD-L1). PD-L1 was evaluated by tumour proportion score (TPS), and PD-L1 expression on at least 1% tumour cell was viewed as positive.
5
Comprehensive Profiling of Tumor-Infiltrating Immune Cells
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TILs and PBMCs were stained for 10-color flow cytometry (detailed antibody list, Supplementary Table 4 ). An extensive literature research focusing on druggable targets was performed to select immune-regulatory molecules. Representative literature and clinical studies underlying the classification in co-inhibitory or co-stimulatory molecules is included in Supplementary Table 1 . Samples were acquired on a Gallios flow cytometer (Beckman Coulter, USA) and analyzed using Kaluza v2.1 (Kaluza, RRID:SCR_016182, Beckman Coulter, USA; gating strategy in Supplementary Fig. 12 ). FFPE sections containing tumor tissue and healthy tissue were selected for each patient. The tumor front was delineated digitally on scanned slides using Aperio ImageScope v12.4.0 (Leica, Germany). Whole section slides and tissue micro arrays (∅ 1.2 mm) of tumor specimens were stained on a Leica BOND-MAX or Roche Ventana platform according to the manufacturer’s instructions (details in Supplementary Table 4 ).
6
Immunohistochemistry Staining Protocols
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All IHC stainings were carried out on immunostaining instruments (Roche Ventana Discovery or LEICA BondMax) following manufacturer’s guidelines. The following antibodies were used in this study: anti-GPR158 (ab121388, Abcam), anti-ATRX (HPA001906, Sigma), anti-IDH1R132H (DIA H09, Dianova), anti-Olig2 (ab33427, abcam), anti-Pdgfrα (ab15501, Abcam), anti-Sox2 (AB 5603, Chemicon) and anti-DCX (ab18732, Abcam).
7
Comprehensive Immunohistochemistry Profile
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Thick sections 4 μm in size were cut from paraffin‐embedded tissue blocks for multiple immunohistochemistry analyses, including TTF‐1 (clone 8G7G3/1, ZECA), Napsin A (polyclonal, MXB), β‐catenin (clone UMAB15,BIO), Chromogranin A (clone EP38, BIO), synaptophysin (polyclonal, MXB), AFP (clone 2ZA06, BIO), and PD‐L1 (clone 22C3, DAKO). All staining was performed on Leica Bond‐Max or Roche Ventana. Omission or replacement of the primary antibody with PBS was routinely used as a negative control.
8
Comprehensive Immunohistochemical Profiling of Tumors
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Four-μm sections cut from representative available FFPE blocks of each case were evaluated for a panel of immunohistochemical markers, including Pan-CK (clone AE1/AE3, BIO), TTF-1 (clone 8G7G3/1, ZECA), EMA (clone GP1.4, BIO), ER (clone SP1, Roche), PR (clone 1E2, Roche), CD34 (clone EP88, BIO), STAT6 (clone, EP325, MXB), S-100 (clone 4C4.9, MXB), SMA (clone UMAB237, BIO), Desmin (clone MX046, MXB). The stain was performed on Leica Bond-Max or Roche Ventana system. The positive percentage and expression pattern (membrane, cytoplasm or nucleus) were evaluated. Staining results were regarded as positive when it expressed moderate or greater intensity in more than 10% of neoplastic cells. Reactivity less than 10% of neoplastic cells were considered as negative.
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