Cd11b

The expression of cell surface markers was identified by the FCM method using CD11b (Abcam, Cambridge, UK), CD44 (Thermo, MA, USA), CD90 (BioLegend, CA, USA), and HLA-DR (BD, NJ, USA). BD FACS Celesta™ flow cytometer (BD, CA, USA) was used for detection. After induced by adipogenic and osteogenic induction solutions respectively for 21 days, the stem cell differentiation ability was detected by Oil Red Assay and Alkaline Phosphatase Assay (KeyGEN, Jiangsu, China).

Mice were trans-cardiacally perfused with 4% paraformaldehyde. Their brains and colons were post-fixed with 4% paraformaldehyde for 4 h, cytoprotected in 30% sucrose solution, freezed, cut using a cryostat (Leica, Nussloch, Germany), and immunostained according to the method of Jang et al. (11 (link)). Briefly, the sections were washed with phosphate buffered saline, blocked with normal serum, incubated with antibodies for Iba1 (1:200, Abcam), LPS (1:200, Millipore), NF-κB (1:100, Cell Signaling), CD11b (1:200, Abcam), CD11c (1:200, Abcam), and/or NeuN (1:200, Millipore) overnight, and treated with the secondary antibodies for 2 h. Secondary antibodies conjugated with with Alexa Fluor 488 (1:200, Invitrogen) or Alexa Fluor 594 (1:200, Invitrogen) were then treated to visualize. Nuclei were stained with 4′,6-diamidino-2-phenylindole, dilactate (DAPI, Sigma). Immunostained tissue slices were scanned with a confocal laser microscope.

Three or more HSV-tk transgenic mice and WT mice were sacrificed starting from the age of 3-month-old, and then every three months until the mice reached an age of over 13 months. Before sacrifice, blood was collected from the orbital plexus for serologic tests of the values of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) using clinical chemistry Analyzer (Abbott Architect c8000, Chicago, IL, USA). The livers were examined for gross lesions, then fixed with 10% buffered formaldehyde (formaldehyde 4%, NaH₂PO₄·H₂O 0.4%, Na₂HPO₄ 1.3%), the liver tissue from each lobe and tumors were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed following the instruction of the procedure for each antibody. Sections prepared from the paraffin-embedded blocks were stained with a CD3 antibody (Beyotime, Shanghai, China) and CD11b (Abcam), both in 1:200. For microarray analysis and immunofluorescence, non-tumorous tissues and individual tumors were snap frozen in liquid nitrogen and stored at −80 °C until used. Histopathological changes in these livers were observed by two pathologists independently.

Paraformaldehyde (4%)-fixed liver tissues were embedded in paraffin, and 5-μm thickness sections were subjected to HE staining, Sirus red staining and IHC staining. Antibodies used in IHC staining included LPC-specific marker CK19 (Proteintech, Peprotech Group, Rocky Hill, NJ, USA), DJ-1 (Abcam, Cambridge, UK), Myeloperoxidase (Biocare Medical, Concord, CA, USA), CD11b (Abcam) and F4/80 (AbD Serotec, Kidlington, UK).

The western blot technique was performed in cerebral cortex tissue lysates, as previously described [28 (link)]. The primary antibodies used were PLP (proteolipid protein), MBP (myelin basic protein), major histocompatibility complex class II (MHC-II), CD11b (Abcam, Cambridge, UK), synaptotagmin, synapsin IIa (BD Bioscience, Madrid, Spain), Tuj-1 (Neuromics, Minneapolis, USA), and caspase 3 (active fragment of 17 kDa) (Cell Signaling, Massachusetts, USA). Membranes were washed, incubated with the corresponding HRP-conjugated secondary antibodies, and developed with the ECL system (ECL Plus; Thermo Fisher Scientific, Illinois, USA). All the membranes were stripped and incubated with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control (Chemicon, California, USA). Band intensity was quantified with the ImageJ 1.44p analysis software (National Institutes of Health, USA). The densitometry analysis is shown in arbitrary units normalized to the loading control. Additional file 1: Table S1 shows that the basal values of the various proteins evaluated by western blotting in the pup’s cortices display no significant differences when comparing both the WT and TLR4-KO groups.