Shandon finesse 325 microtome

Brains were cut to 5 μm sagittal sections using a Shandon Finesse 325 Microtome (Thermo Fisher Scientific) and were stained for Iba1, GFAP or appropriate isotypes and negative controls. Three non-consecutive slides were stained for each cell type from each mouse brain. Blinded slides were sent to the Beatson Institute (University of Glasgow) to be scanned. Iba1 and GFAP hippocampal area quantification was carried out using ImageJ software.

Explanted samples were washed in phosphate-buffered saline and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer–thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Slides were stored at 37^oC overnight to dry completely before staining. Hematoxylin and eosin and Van Gieson’s stainings were performed using a Shandon Varistain 24-4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was performed using a silver plating kit (In Vitro Diagnostic Medical Device) for the detection of microcalcification. Collagen and elastin contents of the explants in the Van Gieson’s staining were quantified using ImageJ software. Results were expressed as proportion of area occupied by collagen or elastin within the graft tissue.

Tissue samples obtained during autopsy were fixed in 5% buffered formalin (pH 7.4), embedded in paraffin and histologically sectioned into 5 µm thick slices using a Shandon Finesse 325 Microtome from Thermo. Standard hematoxylin and eosin staining was performed as described elsewhere⁴⁶ . Images of tissue samples were obtained using a Nikon Eclipse Ti-U inverted optical microscope coupled with a colored camera under a Plan 4 × objective. Image analysis was made using NIS-Elements F 3.0 software.

Study design: This was a laboratory-based retrospective cross-sectional study on archival formalin-fixed paraffin embedded (FFPE) lymphoma tissue stored over a 4 year period from January 2011 to December 2014 in the Histopathology Laboratory of the University Teaching Hospital, in Lusaka. The University Teaching Hospital is a tertiary referral and teaching hospital with a bed capacity of about 2000. It is the biggest reference hospital and the centre for all histopathology diagnostic work in Zambia.
Specimens: The blocks of tissues included cases from all age groups ranging from 9 months to 85 years. The blocks were prepared from tissues obtained from different anatomical sites (anal tissue, right parotid tumour, inguinal lymph node, cervical lymph node, from the nose, ulcerated skin, etc.) from 86 male and 64 female patients.
Histopathology: Tissue sections were cut at 6μm on a Shandon Finesse 325 microtome (Thermo Scientific-Shandon, California, USA). Diagnosis of lymphomas was examined by Haematoxylin and Eosin staining of the histological sections which were examined by an experienced pathologist and grouped as Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL).

Explanted samples (Figure 2C) were washed in PBS and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer-thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Slides were stored at 37°C overnight to dry completely before staining. Hematoxylin and eosin, and Van Gieson’s stainings were performed using a Shandon Varistain 24–4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was carried out using a Silver plating kit (In Vitro Diagnostic Medical Device, Darmstadt, Germany) for the detection of microcalcification. Collagen and elastin contents of the explants in the Van Gieson’s staining were quantified using ImageJ software via color deconvolution, to separate and convert the collagen and elastic fibers to the mean Gray value of the pink and purple and colour intensity, respectively. Threshold values were set for each channel and area-based analysis was used to extract and quantify the regions of interest (ROIs) from the image. Results were expressed as proportion of area occupied by collagen or elastin within the graft tissue.