Representative tumors from each participant were obtained from FFPE archival biopsy samples. The tissue was arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus (Superbiochips Laboratories). Immunohistochemical staining was performed using an automatic immunostainer (DAKO) according to the manufacturer's instructions. Anti-ATM antibody was used as the primary antibody (Y170). Loss of ATM expression was defined as a greater than 80% loss of nuclear staining. Multiplex immunofluorescence was kindly performed by J Tau be at Johns Hopkins University (Baltimore, MD).
Automatic immunostainer
Manufactured by Agilent Technologies
Sourced in Denmark
The Automatic Immunostainer is a laboratory equipment designed for automated immunohistochemical staining of tissue samples. It performs the necessary steps for antigen retrieval, primary antibody incubation, and subsequent detection and visualization of the target proteins in a consistent and efficient manner.
Automatically generated - may contain errors
5 protocols using automatic immunostainer
1
Tissue Array Analysis of ATM Loss
2
Tissue Microarray-Based Immunohistochemistry for FDC Markers
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Tissue microarray (TMA) blocks were created from FDC-S and 72 controls (control group #1, Supplementary Table 3 ), using an automated tissue microarray (TMA Master, 3DHistech, Budapest, Hungary). Three representative tumor cores (1 or 1.5 mm diameter) were identified on hematoxylin and eosin (H&E) stained sections and punched from the original tissue blocks.
Immunohistochemistry for known and novel FDC markers was performed on these TMAs. In order to further confirm their specificity, FDCSP and SRGN were additionally applied on TMAs including 114 carcinomas of different origin, previously described [24 (link)], and on whole tissue sections of 28 additional control cases (control group #2,Supplementary Table 3 ). Antibodies applied are detailed in Supplementary Table 5 .
Immunohistochemistry for FDCSP and SRGN was performed with automatic immunostainer (Dako, Glostrup, Denmark) or manually, showing equivalent results; immunohistochemistry on controls was performed automatically (Leica Biosystems, Wetzlar, Germany).
Stained sections were digitalized using the Aperio Scanscope System (Leica Biosystems) and evaluated by two pathologists (S.H. and L.L.). Cases were scored positive when reaction occurred on at least 30% of tumor cells.
Immunohistochemistry for known and novel FDC markers was performed on these TMAs. In order to further confirm their specificity, FDCSP and SRGN were additionally applied on TMAs including 114 carcinomas of different origin, previously described [24 (link)], and on whole tissue sections of 28 additional control cases (control group #2,
Immunohistochemistry for FDCSP and SRGN was performed with automatic immunostainer (Dako, Glostrup, Denmark) or manually, showing equivalent results; immunohistochemistry on controls was performed automatically (Leica Biosystems, Wetzlar, Germany).
Stained sections were digitalized using the Aperio Scanscope System (Leica Biosystems) and evaluated by two pathologists (S.H. and L.L.). Cases were scored positive when reaction occurred on at least 30% of tumor cells.
3
ATM and PD-L1 Immunohistochemistry Protocol
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Immunohistochemistry (IHC) was performed using an automatic immunostainer (Dako, Glostrup, Denmark) according to the manufacturer’s instructions. To assess ATM protein expression by IHC, a primary anti-ATM antibody was used (Y170; Abcam Plc., Cambridge, UK). Regardless of the cytoplasmic staining status, cancer cells showing nuclear staining were considered positive for ATM. Loss of ATM protein expression was defined as nuclear expression in ≤20% of the stained cells as previously reported.16 (link) For PD-L1 IHC, we used Dako PD-L1 IHC 22C3 pharmDx kit (Agilent Technologies, California, USA) with the EnVision FLEX visualization system (Agilent Technologies) and counterstained with hematoxylin according to the manufacturer’s instructions. PD-L1 protein expression was assessed using the Combined Positive Score (CPS), which refers to the percentage of PD-L1-stained cells (tumor cells, lymphocytes, and macrophages) among viable tumor cells. Specimen was considered PD-L1-positive when CPS was ≥1.
4
Immunocytochemical Analysis of Cell Cultures
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Samples taken from cell culture flasks were retained in PreservCyt™. Subsequently, cytological preparations were obtained in monolayer apparatus with ThinPrep, the first of which was colored by Papanicolaou staining. The others were used for immunocytochemical staining, performed with the avidin-biotin-peroxidase technique in an automatic immunostainer (DakoCytomation, Carpinteria, CA, USA), using the following primary antibodies: cytokeratin AE1/AE3 (dilution 1/5), cytokeratin 18 (dilution 1/2), cytokeratin 19 (dilution 1/100), epithelial membrane antigen (EMA) (dilution 1/100), Ki-67 (dilution 1/100), mitochondria (dilution 1/75), vimentin (dilution 1/2) and the detection system LSAB Plus (DakoCytomation). The sections were incubated with primary antibodies for 16 h at 4°C and then with biotinylated secondary antibodies and avidin-peroxidase for 30 min at 37°C. Detection was done with diaminobenzidine chromogen (DAB) for 20 min at 20°C and nuclear contrast was obtained by immersion for 2 min in Meyer's hematoxylin. The sections were finally mounted with glycerine and special coverslips. At least 3 experiment for each sample were performed.
5
Immunohistochemical Evaluation of PD-L1 in Tumors
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Representative tumors from each participant were obtained from formalin-fixed, paraffin-embedded (FFPE) archival biopsy samples. The tissue was arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus (SuperBioChips Laboratories). Immunohistochemical staining was performed using an automatic immunostainer (Dako) according to the manufacturer's instructions. We used the Dako PD-L1 IHC 22C3 pharmDx Kit (Agilent Technologies) with the EnVision FLEX visualization system and counterstained with hematoxylin according to the manufacturer's instructions. PD-L1 protein expression was determined using CPS, which is the number of PD-L1–stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100. The specimen was considered to express PD-L1 if CPS was ≥1.
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