Bond max

A total of 80 clots were included in this study. Following MT retrieval, thrombus tissues were immediately fixed in 10% NBF and subsequently shipped to a histology core facility for processing. On arrival, clots were randomly assigned to four groups according to planned formalin fixation duration: group 1, 1–30 days (N = 20); group 2, 30–60 days (N = 20); group 3, 60–90 days (N = 20); and group 4, 90+ days, up to 2 years (N = 20). Thrombus tissues were further processed using a previously published, routine lab tissue processing protocol (12 (link)). Samples were embedded in paraffin blocks and cut into 3 μm sections. Serial sections were stained with H&E and Martius Scarlet Blue (MSB) following a lab routine staining protocol as described previously (12 (link), 13 (link)). IHC was also performed for platelets marker (CD42b) on an autostainer (Leica Biosystems Ltd.; Nussloch GmbH, Germany; model Bond Max). Tris-EDTA was used for antigen retrieval. The sections were incubated with the primary antibody anti-CD42b (ab27669, 1:200 dilution) for 20 min. Additionally, negative controls were included and incubated with non-immune serum instead of primary antibody. The RedMap kit (Bond Polymer Refine Red Detection System, Leica Biosystems Ltd.) was used for visualization.
Tissue processing, sectioning, and all stains were performed by the same trained, experienced lab personnel.

Immunoreactions were revealed by Bond Polymer Refine Detection on an automated autostainer (Bond^™ Max, Leica Biosystem, Milan, Italy). ER and PgR expression was evaluated using mAb 6F11 (Leica, Novocastra) and mAb 1A6 (Leica, Novocastra) respectively. Cell proliferation and HER2 overexpression were tested using the Ki-67 mAb (MIB1, Dako, Milan, Italy) and the polyclonal antibody A0485 (Dako), respectively. Tumors were also characterized for cytokeratin 19 expression (Dako). HER2 IHC positivity was defined according to ASCO/CAP guidelines [20 (link)]. ER and PgR were considered positive when >1% and ≥20% of the neoplastic cells showed distinct nuclear immunoreactivity, respectively. Ki-67, based on the median value of our series, was regarded as high if more than 15% of the cell nuclei were immunostained. Evaluation of the IHC results, blinded to all patient data, was performed independently and in blinded manner by two investigators (SB and MM).

Tissues have been fixed in neutral tamponed formalin 10% and included in paraffin. They have been cut in 4 µm sections with a Microm HM 355S (Thermo Fisher Scientific, Waltham, USA). Mouse monoclonal antibody VE1, was used to detect the BRAF^V600E mutation. Stainings were performed with Bond-Max (Leica Biosystems). Antigen retrieval was performed during 60 min at 96 °C in pH9 buffer Bond Epitope Retrieval Solution 2 (Leica Biosystems). VE1 hybridoma supernatant was diluted one-third and incubated at 37 °C for 32 min. Staining was revealed with bond polymer refine red detection kit (Leica Biosystems).
The same procedure was carried out for the other commercial antibodies against: CD14, CD1a, CD68/PGM1, CD207, S100, p16, Ki67, and TNFα.

Immunohistochemical staining was carried out using the Bond‐Max (Leica Biosystems), an automated slide staining system. Pretreatment was done with ER2 (Leica Biosystems) for 20 min for both SMARCA4 and SMARCE1. A monoclonal mouse antibody against SMARCA4/ BRG1 (clone E8V5B, Cell Signaling Technology) applied in a 1:400 dilution and a monoclonal rabbit antibody, directed against SMARCE1/BAF57 (clone E6H5J, Cell Signaling Technology) applied in a 1:300 dilution were used. Immunostaining was visualized with the Polymer Refine Detection Kit (Leica Biosystems). Hemalaun served as counterstaining.

CD3 (Dako A0452), PHOX2B (Abcam ab183741), and HLA-ABC (Abcam ab70328) antibodies were used to stain formalin fixed paraffin embedded tissue slides. Staining was performed on a Bond Max automated staining system (Leica Biosystems). The Bond Refine polymer staining kit (Leica Biosystems, DS9800) was used. The standard protocol was followed with the exception of the primary antibody incubation which was extended to 1 h at room temperature. CD3, PHOX2B and HLA-ABC antibodies were at 1:100, 1:500 and 1:1,200 dilutions, respectively. Antigen retrieval was performed with E1 (Leica Biosystems) retrieval solution for 20 min (E2 for PHOX2B). Slides were rinsed, dehydrated through a series of ascending concentrations of ethanol and xylene, then coverslipped. Stained slides were then digitally scanned at 20× magnification on an Aperio CS-O slide scanner (Leica Biosystems).