Eg1150 embedding center

All tissues were embedded in paraffin by the Van Andel Institute Pathology and Biorepository Core following internal standard operating procedures. Upon receipt, tissues were dehydrated and cleared using a Tissue-Tek VIP 5 (Sakura) and an automated protocol consisting of 60’ in 70% ethanol; 60’ in 80% ethanol; 2 x 60’ in 95% ethanol; 3 x 60’ in 100% ethanol; 2 x 30’ in xylene; and 1 x 30’ and 1 x 45’in paraffin. Tissues were embedded in paraffin with a Leica EG1150 embedding center. Three 5-µm thin sections spaced 150 µm apart were cut from each tissue for hematoxylin and eosin (H&E) staining using a Leica rotary microtome. The remaining tissue was conserved as a paraffin embedded tissue block. H&E staining was performed with a Tissue-Tek Prisma Plus Automated Slide Stainer (Sakura) and Tissue-Tek Prisma H&E Staining Kit #1.

The serum‐free conditioned medium from the different cell lines was precooled and mixed with Matrigel at a ratio of 1:1. A volume of 200 µl of the conditioned medium was subcutaneously injected into BALB/c athymic nude mice. Matrigel plugs were maintained for 10 days in the mice and then fixed with 4% PFA immediately followed by embedding in paraffin with a Leica EG1150 embedding center. Five‐micron sections were prepared with a Leica RM2235 microtome, deparaffinized using a Leica Multistainer, and subjected to antigen retrieval using 0.1 m sodium citrate solution. The sectioned Matrigel plugs were immunofluorescence stained with antibodies of macrophage markers F4/80, CD204, and CD206. The fluorescence images were obtained by using a confocal laser‐scanning microscope (Carl Zeiss LSM710, Germany).