Kaiser s glycerin gelatine

Microscope slides with 3 μm sections from paraffin-embedded tissue were dewaxed and rehydrated following standard procedures (preheating at 60°C, xylene substitute [Neo-Clear, Merck KgaA, Darmstadt, Germany] graded series of ethanol (100%, 96%, 80% and 70%), followed by double distilled water). After antigen demasking (microwave irradiation at 600 W, 0.1 M citrate buffer pH 6.0) and overnight incubation with the primary antibodies (anti-ß₃-integrin antibody Abcam ab179473, 1:500, anti-CD31 antibody Abcam ab28364 1:50, anti-Ki-67 antibody SP6 Abcam ab16667 1:100 [all Cambridge, United Kingdom]) at 4°C, tissue samples were further processed using the EnVision+ System HRP (DAB or AEC) (DAKO Diagnostika, Hamburg, Germany) kit according to the manufacturer´s instructions. Slides were counterstained with Mayer´s Haemalaun (Merck KgaA, Darmstadt, Germany) and covered with Kaiser´s Glycerin Gelatine (Merck KgaA, Darmstadt, Germany). The optical density of ß₃-integrin expression was measured in ten random fields at 200x magnification using ImageJ (“Fiji” version, www.fiji.sc). CD31-positive microvessels and Ki-67-positive nuclei were quantified in ten random high-power fields at 200x magnification.

Three micrometer tissue sections were cut from the formalin-fixed and paraffin-embedded tumor tissue and stained with regard to microvascular density (CD31) and tumor cell proliferation (Ki-67). After de-waxing and rehydration following standard procedures (pre-heating at 60 °C, washing in xylene substitute (Neo-Clear, Merck KgaA, Darmstadt, Germany) and rehydration in a graded series of ethanol (100, 95, 80, and 70%, respectively) followed by double distilled water, antigen demasking was performed by microwave irradiation at 600 W in a 0.1 M citrate buffer solution (pH 6.0). Tissue samples were subsequently antibody-incubated at 4 °C over night (antibodies: monoclonal rabbit anti-Ki-67 antibody; SP6, Abcam ab16667 1:100, Cambridge, United Kingdom; polyclonal rabbit anti-CD31 primary antibody; Abcam ab28364 1:50, Cambridge, United Kingdom).
According to the manufacturer’s instructions, further work-up of tissue samples was performed using the EnVision + System HRP (DAB or AEC) (DAKO Diagnostika, Hamburg, Germany) kit. Slides were counterstained using Mayer’s Haemalaun (Merck KgaA, Darmstadt, Germany) and covered with Kaiser’s Glycerin Gelatine (Merck KgaA, Darmstadt, Germany). Results were quantified as the number of positively stained nuclei (Ki-67) or positively stained microvessels (CD31) in ten random fields at 200× magnification.

For immunohistochemical assessment of tumor microvascular density, tumor sections were incubated with a polyclonal rabbit anti-CD31 primary antibody (Abcam ab28364 1:50, Cambridge, United Kingdom) overnight. Further work-up of tissue samples was performed using the EnVision+ System HRP (AEC) (DAKO Diagnostika, Hamburg, Germany) according to the manufacturer´s instruction. Counterstaining was performed using Mayer´s hemalum (Merck Millipore, Darmstadt, Germany) and slides were covered with Kaiser´s Glycerin Gelatine (Merck Millipore, Darmstadt, Germany). Tumor microvessels were quantified as the average number of endothelial cells in 10 random fields at 200x magnification [32 (link)].