Serial sections (4 μm) were cut from the block of paraffin-embedded tissue, stained with hematoxylin and eosin, and used for the following immunohistochemical stains: CD20 (L26 [1:400]; DAKO, Glostrup, Denmark), CD3 (LN10 [1:200]; Novocastra, Newcastle, UK), CD5 (4c7 [1:50]; Novocastra), CD10 (56C6 [100:1]; Novocastra), CyclinD1 (SP4 [1:50]; Nichirei, Tokyo, Japan), Ki-67 (MIB-1 [1:2500]; Novocastra), IgG (polyclonal [1:20,000]; DAKO), and IgG4 (HP6025 [1:10000]; The Binding Site, Birmingham, UK). Following immunostaining using an automated Bond Max stainer (Leica Biosystems, Melbourne, Germany), the numbers of IgG4+ and IgG+ cells were estimated in areas with the highest density of IgG4+ cells. In accordance with the consensus statement on the pathological features of IgG4-RD17 (link), three different high-power fields (HPFs) (total magnification, ×400) were examined to calculate the average number of IgG4+ cells per HPF and the IgG4+/IgG+ cell ratio. In situ hybridization was also performed for κ and λ-light chains (Leica Biosystems) using a Bond Max stainer.
Bond max stainer
Manufactured by Leica
Sourced in Germany, Australia
The Leica BOND-MAX stainer is an automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining system. It is designed to provide consistent and reliable staining results for a wide range of antibodies and probes used in clinical and research laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Bond polymer refine detection kit, by Leica (2 mentions)
κ and λ light chains, by Leica (1 mentions)
Cd5 4c7, by Leica (1 mentions)
Cd10 56c6, by Leica (1 mentions)
Cyclin d1, by Nichirei Biosciences (1 mentions)
Lmp 1, by Leica (1 mentions)
Inform eber, by Leica (1 mentions)
Tia 1, by Leica (1 mentions)
Ab86809, by Abcam (1 mentions)
P16 clone jc8, by Santa Cruz Biotechnology (1 mentions)
Ventana benchmark stainer, by Roche (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Mmp9 antibody, by Abcam (1 mentions)
D5j4e, by Cell Signaling Technology (1 mentions)
Clone d4d6, by Cell Signaling Technology (1 mentions)
Cd30 clone berh2, by Agilent Technologies (1 mentions)
Hamamatsu nanozoomer, by Hamamatsu Photonics (1 mentions)
Cd68 clone pg m1, by Agilent Technologies (1 mentions)
Cd20 clone l26, by Agilent Technologies (1 mentions)
Tissuestudio 64, by Definiens (1 mentions)
24 protocols using bond max stainer
1
Immunohistochemical Analysis of IgG4-RD
2
Immunohistochemical Analysis of Lymphoma Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specimens were fixed in 10% formaldehyde and embedded in paraffin. Three‐micrometer‐thick sections were cut from the paraffin‐embedded tissue blocks and stained with hematoxylin and eosin.
Paraffin sections of each tissue sample were used for immunohistochemical staining with antibodies to CD3 (clone: LN10, 1:200; Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), CD5 (clone: 4C7, 1:100; Novocastra Laboratories, Ltd.), CD10 (clone: 56C6, 1:100; Novocastra Laboratories, Ltd.), CD15 (clone: Carb‐3, 1:50; DAKO, Glostrup, Denmark), CD20 (clone: L26, 1:100; DAKO), CD30 (clone: Ber‐H2, 1:40; DAKO), EBV nuclear antigen 2 (EBNA2) (clone: PE2, 1:100; Abcam, Cambridge, MA, USA), EBV latent membrane protein 1 (LMP‐1) (clone: CS1‐4, 1:50; Novocastra Laboratories, Ltd.), and Ki‐67 (clone: MIB‐1, 1:2500; DAKO). Staining was performed using the automated Bond Max Stainer (Leica Biosystems, Wetzlar, Germany).
The EBV was detected by in situ hybridization for EBER using the automated Bond Max Stainer (Leica Biosystems).
Paraffin sections of each tissue sample were used for immunohistochemical staining with antibodies to CD3 (clone: LN10, 1:200; Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), CD5 (clone: 4C7, 1:100; Novocastra Laboratories, Ltd.), CD10 (clone: 56C6, 1:100; Novocastra Laboratories, Ltd.), CD15 (clone: Carb‐3, 1:50; DAKO, Glostrup, Denmark), CD20 (clone: L26, 1:100; DAKO), CD30 (clone: Ber‐H2, 1:40; DAKO), EBV nuclear antigen 2 (EBNA2) (clone: PE2, 1:100; Abcam, Cambridge, MA, USA), EBV latent membrane protein 1 (LMP‐1) (clone: CS1‐4, 1:50; Novocastra Laboratories, Ltd.), and Ki‐67 (clone: MIB‐1, 1:2500; DAKO). Staining was performed using the automated Bond Max Stainer (Leica Biosystems, Wetzlar, Germany).
The EBV was detected by in situ hybridization for EBER using the automated Bond Max Stainer (Leica Biosystems).
3
Immunohistochemical Analysis of Submandibular Gland Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All of the diseased and normal tissue samples used in this study were surgically resected specimens of the submandibular glands. The specimens were fixed in 10% formaldehyde and embedded in paraffin. Serial 4-μm-thick sections were cut from the blocks of paraffin-embedded tissues and stained with hematoxylin and eosin (H&E). The sections were immunohistochemically stained using an automated Bond Max stainer (Leica Biosystems; Wetzlar, Germany). The following primary antibodies were used: IL-13 (2B5; 1:300; Abnova; Taipei City, Taiwan), c-kit/CD117 (YR145; 1:100; EPITOMICS; Burlingame, CA, USA), IgG (polyclonal; 1:20,000; Dako; Glostrup, Denmark), and IgG4 (HP6025; 1:400; The Binding Site; Birmingham, UK).
4
FNA Cytology and Ki-67 Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FNA cytology was performed and evaluated as part of the routine clinical workup. The aspirated material was mainly used for cytomorphological evaluation. In a subgroup of patients, a part of the aspirate was used to determine Ki-67 proliferation index by immunocytochemistry. In short, air-dried smears were fixed in buffered 4% formaldehyde solution followed by methanol and acetone. The monoclonal Ki-67 antibody (clone MIB-1, DAKO M7240) was used with a dilution of 1:200. In Cohort A, prior to 2010, the smears were manually stained with immunoperoxidase-avidin-biotin technique. Since 2010 the staining has been performed by an automated BOND-MAX stainer (Leica Biosystem, Germany) with standardized methodology with BOND polymer refine detection kit, poly-HRP (horse-radish-peroxidase) reagent and diaminobezidin (DAB). Scoring was performed by calculating the percentage of positive cells (brown stained nuclei) by counting at least 200 tumor cells. Analyses of reactive lymph nodes were included as positive control which revealed distinct nuclear staining and omission of the primary antibody served as negative control. In Cohort B the immunological technique was performed as previously described by Sofiadis et al. [9 (link)].
5
Immunohistochemistry Protocol for Lymphoma Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were fixed in 10% formalin and embedded in paraffin. An automated BOND-MAX stainer (Leica Biosystems, Melbourne, Vic., Australia) was used to stain the paraffin sections. The following primary antibodies were used (clone, dilutions): CD20 (L26, 1:200), CD3 (LN10, 1:200), CD5 (4C7, 1:100), CD7 (56C6, 1:50), CD8 (C8/144B, 1:100), CD4 (1F6, 1:40), TIA-1 (2G9, 1:500; Beckman Coulter, Brea, CA, USA) and GranzymeB (GrB-7, ready to use) (Nichirei, Tokyo, Japan). Primary monoclonal antibodies raised against the human TCRβ (βF1; TCR1151, 8A3, 1:50 [Thermo Scientific, Waltham, MA, USA]) and TCR-γ chain constant region (TCR1153, γ3.20, 1:80 [Thermo Scientific]) were used to detect the αβ and γδ TCR, respectively.(12 (link)) In situ hybridization with Epstein–Barr virus (EBV)-encoded small RNA (EBER) probes (INFORM EBER; Leica Biosystems) was used to detect EBV.
The samples of CD20, CD3, CD10, CD5, CD7, CD8, CD4, TIA-1,(13 (link)) granzyme B(14 (link),15 (link)) and EBER antigens were scored as positive when ≥30% of the lymphoma cells were positively stained. The samples of TCR γ, TCR δ and TCR β antigens were scored as positive when ≥50% of the lymphoma cells were positively stained.
The samples of CD20, CD3, CD10, CD5, CD7, CD8, CD4, TIA-1,(13 (link)) granzyme B(14 (link),15 (link)) and EBER antigens were scored as positive when ≥30% of the lymphoma cells were positively stained. The samples of TCR γ, TCR δ and TCR β antigens were scored as positive when ≥50% of the lymphoma cells were positively stained.
6
Immunohistochemical Analysis of SOX4 and p16 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specimens were fixed in 10% formaldehyde and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin-embedded tissue blocks and stained with hematoxylin and eosin. Paraffin sections of each tissue sample were used for immunohistochemical staining with antibodies to SOX4 (polyclonal antibody, ab86809, dilution 1:60; Abcam, Cambridge, MA, USA) and p16 (clone: JC8, dilution 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Immunohistochemical staining was performed using the automated Bond Max Stainer (Leica Biosystems, Wetzlar, Germany).
7
Gastric Mucosal Histology in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three biopsy specimens were obtained from the greater curvature of the antrum, the lesser curvature of the corpus, and the greater curvature of the corpus. The gastric mucosa samples were evaluated according to the updated Sydney system, with the degree of inflammation (mononuclear cell infiltration), neutrophil activity, atrophy, and IM classified and scored as ‘normal,’ 0; ‘mild,’ 1; ‘moderate,’ 2; and ‘marked,’ 3; according to a visual analogue scale (14 (link)). In Group B (control group), biopsy specimens were obtained at every follow up endoscopy, and the newest data was enrolled. In Group A (cancer group), biopsy specimens were obtained at the time of preoperative endoscopy (approximately 1-2 months before ESD).
Furthermore, we conducted a pilot study using an immunohistochemical analysis to elucidate the type of mononuclear cell infiltration. Sections were immunohistochemically stained using an automated Bond Max stainer (Leica Biosystems, Melborne, Australia). The following primary antibodies were used: CD79a (JCB79a, dilution 1:50; Dako, Glostrup, Denmark), CD3 (LN10, dilution 1:100; ABCAM, Cambridge, UK).
Experienced pathologists from Okayama University Hospital performed the histological evaluations. We compared the scores from the histological evaluations of the gastric mucosa between the two groups.
Furthermore, we conducted a pilot study using an immunohistochemical analysis to elucidate the type of mononuclear cell infiltration. Sections were immunohistochemically stained using an automated Bond Max stainer (Leica Biosystems, Melborne, Australia). The following primary antibodies were used: CD79a (JCB79a, dilution 1:50; Dako, Glostrup, Denmark), CD3 (LN10, dilution 1:100; ABCAM, Cambridge, UK).
Experienced pathologists from Okayama University Hospital performed the histological evaluations. We compared the scores from the histological evaluations of the gastric mucosa between the two groups.
8
Immunohistochemical Evaluation of Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The percentage of immunostained cells was evaluated by a specialist in cytology (ET) blinded to the treatment given. Cytospin slides were stained for the nuclear antigen Ki-67. The Ki-67/MIB-1 monoclonal antibody reacts with a human nuclear antigen, which is present in proliferating cells but absent in quiescent cells. Cell cycle analysis shows that the antigen is expressed in the phases of G1, S, G2 and mitosis [19] . The immunostaining was performed with monoclonal antibody Ki67 (DAKO M7240) by an automated BOND-MAX STAINER (Leica Biosystem, Germany) using an avidin-biotin peroxidase system, modified for the cytospin technique. On average 150-200 cells were counted per slide. We considered samples obtained by FNA to be assessable only if they contained intact epithelial cells.
9
Immunohistochemical Analysis of XIAP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was performed on TMA slides. Tumor cell XIAP was detected using a polyclonal rabbit anti-XIAP antibody (ab21278: dilution 1:1000) on Leica BOND-MAX stainer (Leica Biosystems, Germany) according to the manufacturers’ protocol.
We correlated the XIAP results with previously collected and described data like T-cell inflammation of the tumor microenvironment and different molecular tumor cell alterations like TP53, ARIDa1 loss and ERBB2- amplification [17 (link), 18 (link)].
We correlated the XIAP results with previously collected and described data like T-cell inflammation of the tumor microenvironment and different molecular tumor cell alterations like TP53, ARIDa1 loss and ERBB2- amplification [17 (link), 18 (link)].
10
Integrin alpha V Immunohistochemistry Scoring
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) was performed on TMA slides using the Integrin alpha V rabbit monoclonal antibody (ab150361; dilution 1:300; Abcam, UK). Staining and scoring procedures were conducted as previously described12 (link),18 (link)–20 (link). All immunohistochemical stainings were performed using the Leica BOND-MAX stainer (Leica Biosystems, Germany) according to the protocol of the manufacturer.
The membraneous staining pattern was scored manually and independently by two pathologists (A.Q. and H.L.) according to a 4-tier-scoring system. Score 3 + was defined as a strong staining of ≥ 30% of tumor cells or moderate staining ≥ 70%. A weak staining in > 70% or moderate staining in > 30 and ≤ 70% or as strong staining in ≤ 30% of tumor cells was considered as Score 2 + . Score 1 + was assigned when ≤ 70% of tumor cells were weakly positive or ≤ 30% were moderately stained. Less staining was defined as negative (Score 0). Discrepant results were resolved by consensus review.
Expression of Integrin alpha V was correlated with molecular markers including analysis of TP53, Her2/neu, c-myc, GATA6, PIK3CA- and KRAS amplification. A detailed description of the analysis of TP53, KRAS, PIK3CA, Her2/neu and GATA6 is already published11 (link),12 (link),19 (link),21 (link).
The membraneous staining pattern was scored manually and independently by two pathologists (A.Q. and H.L.) according to a 4-tier-scoring system. Score 3 + was defined as a strong staining of ≥ 30% of tumor cells or moderate staining ≥ 70%. A weak staining in > 70% or moderate staining in > 30 and ≤ 70% or as strong staining in ≤ 30% of tumor cells was considered as Score 2 + . Score 1 + was assigned when ≤ 70% of tumor cells were weakly positive or ≤ 30% were moderately stained. Less staining was defined as negative (Score 0). Discrepant results were resolved by consensus review.
Expression of Integrin alpha V was correlated with molecular markers including analysis of TP53, Her2/neu, c-myc, GATA6, PIK3CA- and KRAS amplification. A detailed description of the analysis of TP53, KRAS, PIK3CA, Her2/neu and GATA6 is already published11 (link),12 (link),19 (link),21 (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!