Wright s stain

Fresh tissue from a representative area of the tumors was received and analyzed cytogenetically as part of our diagnostic service. The samples were disaggregated mechanically and enzymatically with collagenase II (Worthington, Freehold, NJ, USA). The resulting cells were cultured and harvested using standard techniques. Chromosome preparations were G-banded with Wright's stain (Sigma-Aldrich; St Louis, MO, USA) and examined. Metaphases were analyzed and karyograms prepared using the CytoVision computer assisted karyotyping system (Leica Biosystems, Newcastle, UK). The karyotypes were described according to the International System for Human Cytogenetics Nomenclature [13 ].
Interphase and metaphase FISH analyses were performed for both cases. The RB1 deletion probe, purchased from Cytocell (http://www.cytocell.co.uk), was used in order to detect deletion of the RB1 locus in 13q14.2. It consists of a 318 kb red probe spanning the RB1 gene and a 13qter green probe acting as a control for chromosome 13. Fluorescent signals were captured and analyzed using the CytoVision system from Leica Biosystems (http://www.leicabiosystems.com/pathology-imaging/cytogenetics/).

The cell line was subjected to chromosome analysis by G-banding. Cells were trypsinized with trypsin/EDTA after treatment with colchicine (colcemid 10 µg/mL) for 1–3 h, and the slides were prepared according to standard methods. Briefly, cells were incubated in 0.075 M hypotonic KCl solution for 20 min and fixed in glacial acetic acid-methanol (3:1). G-banding was performed using 2 × SSC at 65 °C for 2 min and Wright’s stain for 2 min (all reagents were from Sigma-Aldrich, St. Louis, MO, USA). Metaphase images were acquired using an Olympus BX61 microscope (Olympus Corporation, Tokyo, Japan) and analyzed by CytoVision software 7.2 (Leica Biosystems, Newcastle Ltd., UK). A mid-range resolution of 300 bands was achieved. The anomalies were described according to the International System for Human Cytogenetic Nomenclature, 2016 [26 ].

Yolk sacs were fixed in methylcellulose and observed under a stereomicroscope (Nikko ECLIPSE TS100). The survival of embryos was determined by heartbeat or embryo dissolution. The PECAM-1 staining was performed using monoclonal antibody MEC13.3 and detected using an HRP Detection Kit (BD Bioscience). Hemoglobin was stained with o-dianisidine (Sigma-Aldrich). The erythrocytes from the yolk sacs were stained using Wright’s stain (Sigma-Aldrich) and photographed under a microscope (Olympus BX61TRF).

Fresh tissue from a representative area of the tumors was mechanically and enzymatically disaggregated with collagenase II (Worthington, Freehold, NJ, USA). The cells were cultured and harvested using standard techniques [13 ]. Chromosome preparations were G-banded with Wright’s stain (Sigma-Aldrich, St Louis, MO, USA). Metaphases were analyzed, and karyograms were prepared using the CytoVision computer-assisted karyotyping system (Leica Biosystems, Newcastle, UK). The karyotypes were written according to the International System for Human Cytogenomic Nomenclature [14 ].

Packed cell volume (PCV) was determined by the standard microhematocrit technique. The blood was mixed thoroughly and drawn into a haematocrit tube of about ¾ lengths. The free end was flame sealed, and subsequently, the haematocrit tubes were centrifuged in a microhematocrit centrifuge (Haematokrit 20; HettichZentrifugen, Tuttlingen, Germany) at 10,000 rpm for 5 min. The PCV was read directly from the microhematocrit reader and recorded. Total erythrocyte counts were obtained by manual counting, using a hemocytometer set (Hawskley B.S.748, London, UK). Leucocyte count was obtained by automatic counting using a Haematology Analyser (CELL-DYN 3700) as described by [15 (link)]. A refractometer was used to determine the plasma protein in the haematocrit tube, and the values were expressed as g/L. Blood smears were stained with Wright’s stain (Sigma^®, St. Louis, MO, USA) to record the morphology of erythrocytes and leukocytes. The differential leukocyte count was obtained from the microscopic evaluation of stained blood smears using a light microscope (Lieca^® DME, Wetzlar, Germany) under oil immersion (×100). The battlement counting method was consistently used to obtain representative values from the blood smear.

Thin blood film slides were prepared for each of the three camel samples in the four solutions at each of the six incubation periods using ~20 μl of solution. Blood films were prepared using a clean spreader slide that was held at a 45° angle toward the drop of blood on the specimen slide. The sample spread along the edge of the slide by capillary action. The spreader slide was then pushed forward rapidly. The slides were then dried and fixed in methanol (for three minutes) to preserve the morphology of cells. Cells were then stained using Wright’s stain (Sigma-Aldrich, USA) for ~12 min then rinsed with distilled water. Cover slips were mounted over the blood films using DPX mountant and placed on a hot plate for two minutes. The blood film slides were examined under a compound light microscope (CH30RF200, Olympus Optical, Japan) at 100X power oil immersion. The blood film slides were imaged using the Leica DM500 Digital Microscope (Leica, Germany) at the Nanoscopy Center of Kuwait University.