Vectra3 slide scanner

After topical tissue staining and DDSI was completed, each specimen was placed in 10% formalin (Biochemical Science Inc, Swedesboro, NJ) for at least 24 hrs and then prepared for IHC staining of HER2. Four micron slices were cut and mounted on Leica Bond Plus Slides (Cat # 00270) and air-dried at room temperature. Using the automated protocol of the Leica Bond Rx Automated Stainer (Leica Products/Equipment, Leica Microsystems, Inc., Buffalo Groove, IL), the slides were baked for 30 minutes and dewaxed with Leica Bond Dewax solution (Cat #AR9222). The antigen retrieval was Bond Epitope Retrieval 2 (Cat #ar9640), carried out in a pH 9.0 solution for 20 minutes. The HER2 primary antibody dilution was 1:300 for 15 minutes (Abcam Cat # ab16901; Abcam Inc., Cambridge, MA). Primary antibody binding was visualized using Leica Bond Refine Detection kit (Cat # DS9800) with a diaminobenzidine (DAB) chromogen and a hematoxylin counterstain. Bright field, whole fit images of stained tissue (H&E and HER2 IHC) were scanned using the PerkinElmer Vectra3 slide scanner.

Shortly after the 24-h MRI-PAFT scans were acquired, animals were euthanized and brain tissues harvested, frozen and later fixed in preparation for pathological analyses. Several 4-μm-thick sections from each specimen were mounted on slides and stained with either H&E or processed for standard IHC staining of EGFR. For the latter, the primary antibody, Abcam #AB52894 (Abcam Inc., Cambridge, MA) was visualized using Leica Bond Refine Detection kit (Cat # DS9800) with a diaminobenzidine (DAB) chromogen and a hematoxylin counterstain. Both H&E and EGFR IHC slides were scanned using a PerkinElmer Vectra3 slide scanner. Estimates of anti-EGFR staining were determined from 20X magnification IHC images using ImageJ (three sites per tumor). The H DAB vector in the Color Deconvolution 1.7 plugin was applied to each image site to deconvolve the EGFR-stain (brown) from the DAB stain. Next, mean intensity values of the EGFR stain channel were computed from all pixels that met a minimum signal intensity criterium determined from the color images. Mean and standard deviation values were then computed for each of the four animal groups.

Slides are cut at 4 mm and air dried at RT before baking at 60 °C for 30 min. Automated protocol performed on the Leica Bond Rx (Leica Biosystems) includes paraffin dewax, antigen retrieval, peroxide block and staining. Heat induced epitope retrieval using Bond Epitope Retrieval 2, pH9 (Leica Biosystems, AR9640) was incubated at 100 degrees Celsius for 20 min (for anti-cytokeratin 5, Bond Epitope Retrieval 1, pH6.0 (Leica Biosystems, AR9961) was used instead). Primary antibody anti-ER (Cell Marque, 249-R-15-ASR, clone: SP1, 1:35 dilution), anti-p63 (Biocare Medical, CM163B, clone:4A4, 1:200 dilution), and anti-cytokeratin 5 (Abcam, ab236216, clone: SP27, 1:100 dilution) was applied and incubated for 15 min at room temp. Primary antibody binding is detected and visualized using the Leica Bond Polymer Refine Detection Kit (Leica Biosystems DS9800) with DAB chromogen and Hematoxylin counterstain. Slides were imaged using the PerkinElmer Vectra3 slide scanner, and PhenoChart. Staining was qualified and quantified by a breast pathologist (KM).