For characterization of inflammatory cells recruited to the cornea, immunohistochemistry was performed on corneal whole mounts with the macrophage markers F4/80 (AbD Serotec).31 For identification of the intracorneal source of the ECM protein‐TGFBIp, immunohistochemistry of both TGFBIp (1:100 dilution; Abcam) and the macrophage markers F4/80 (1:100 dilution; AbD Serotec) mentioned above was performed on whole mounts of cornea at 7 days after corneal suture placement. The whole mounts of cornea were evaluated using confocal microscopy (Leica TCS‐SP8). Macrophage recruitment was also evaluated as previously described.32 Briefly, 10 areas (eight from the periphery and two from the centre) of each sample were randomly picked and examined under an epifluorescence microscope, and the total numbers of F4/80‐positive cells were counted throughout the whole thickness of the selected area.
F4 80
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Germany, Canada, Japan
F4/80 is a mouse protein that is a member of the EGF-like module-containing mucin-like hormone receptor-like (EMR) family. It is primarily expressed on the surface of macrophages and is commonly used as a marker for the identification and isolation of these cells.
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Lab products found in correlation
α sma, by Merck Group (16 mentions)
Cd11b, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
α sma, by Abcam (7 mentions)
Cd11b, by BD (7 mentions)
Cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Cd11b, by Bio-Rad (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Mcp 1, by Santa Cruz Biotechnology (4 mentions)
P stat3, by Cell Signaling Technology (4 mentions)
Mmp 9, by Abcam (4 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (4 mentions)
Arginase 1, by Santa Cruz Biotechnology (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Collagen 1, by Southern Biotech (3 mentions)
Facscanto, by BD (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Collagen 4, by Abcam (3 mentions)
201 protocols using f4 80
1
Corneal Inflammation Analysis via Immunohistochemistry
2
Histological Analysis of Immune Cell Infiltration
3
Multimodal Imaging of Liver Pathologies
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Masson’s Trichrome and immunohistochemistry was done as described before21 (link),22 (link). For immunofluorescence staining, liver sections were blocked (1% bovine serum albumin in 0.5% PBS-TritonX) and incubated in primary antibodies: F4/80 (AbD Serotec), GFP (Rockland), and BrdU (Cell Signaling). The primary antibodies were detected using AF488 (Vector) and AF647 (Vector). Proliferation in hepatic cells was measured using BrdU uptake. Mice received an IP injection of BrdU 24 h prior to death (50 mg/kg of animal weight).
4
Immunofluorescence Staining of Cryosections
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For immunofluorescence staining, cryosections were fixed in 4% PFA for 10 min, rinsed in phosphate-buffered saline (PBS)-Tween for 10 min and blocked with 10% goat serum for 1 h. Incubation with primary antibodies CD11b (BD Pharmingen, Heidelberg, Germany), F4/80 (MCA497, Serotec, Raleigh, NC, USA), pH2AX (Cell Signaling) and DHE (Invitrogen, Grand Island, NY, USA) was performed at 4 °C overnight. Slides were rinsed in PBS and incubated with appropriate fluorescence-labeled secondary antibodies (AlexaFluor 488 and 564, Invitrogen) for 1 h at room temperature. Slides were washed twice in TBS-Tween and mounted with Vectashield containing DAPI (Linaris, Wertheim, Germany). All sections were analyzed and documented using an Imager Z1 fluorescence microscope together with Axiovision software (Carl Zeiss, Jena, Germany).
5
Profiling Adipocyte-Derived Microvesicles
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Ultracentrifuge purified MPs from 3T3-L1 adipocytes treated with palmitic acid or control were intraperitoneally injected into 8 weeks old C57BL/6 mice. After three or four days, cells infiltrated in the peritoneal cavity were collected with 5 ml x 2 of cold PBS. After removing red cells with RBC lysis buffer, isolated cells were incubated with CD45 (BD Pharmingen), CD11b (eBioscience), F4/80 (AbD Serotec, Raleigh, NC), and Ly6G (clone 1A8) (Biolegend, San Diego, CA) for 30 min on the ice, washed, and analyzed by flow cytometry (BD LSRII Flow Cytometer System).
6
Quantifying Immune Cell Infiltration in Mouse Brain Injury
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C57Bl/6 mice were euthanized at 7 days post-injury and transcardially perfused with PBS, followed by 4 % paraformaldehyde. Brains were removed, post-fixed, and cryoprotected in 30 % sucrose. Thirty-micrometer sections were cut on a cryostat (Microm HM550, Thermo Fisher Scientific). Brain sections were treated with blocking buffer (10 % goat serum/0.1 % BSA/0.01 % Triton X-100) for 1 h at RT and stained O/N at 4 °C with antibodies against CD45-FITC (1:200, eBioscience, San Diego, CA, USA), CCR2 (1:100, Abcam, Cambridge, MA, USA), CD11b (1:200, Millipore, Darmstadt, Germany), and F4/80 (1:200, AbD Serotec, Raleigh, NC). After washing with PBS, three times, secondary Abs (1:200; anti-Rabbit-Cys3; Jackson ImmunoLaboratories, West Grove, PA, USA, anti-Hamster rat-Cys3; Jackson ImmunoLaboratories) were added and incubated for 1 h at RT. Slides were washed with PBS for 5 min, three times, and then coverslipped with mounting media with DAPI (Prolong Gold with DAPI, Invitrogen). The stained brain tissue sections were photographed with a ×20 objective using the BIOREVO all-in-one fluorescence microscope (BZ-9000 Generation II, Keyence microscope), and positive signal was measured using BZ-9000 Generation II analyzer (Keyence).
7
Liver Tissue Analysis Protocol
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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).
8
Evaluating Cell Death and Proliferation in Colon Tissue
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Colon tissue was fixed overnight in 4% paraformaldehyde, embedded in paraffin, and 4 μm sections were made. Cell death was evaluated on paraffin sections by TUNEL staining (DeadEnd Fluorometric TUNEL System, Promega) according to the manufacturer's instructions. For quantification, the numbers of TUNEL-positive cells in at least three different 200× magnification fields from three different cross-sections each were counted per mouse. For assessing cell proliferation, mice were injected intraperitoneally with 100 mg/kg BrdU 2 h before sacrifice. Immunostaining was performed with anti-BrdU (Chemicon). For quantification, the numbers of BrdU-positive cells in all well-oriented and longitudinally sectioned crypts in at least three different 200× magnification fields from three different cross-sections each per mouse were counted. For staining of macrophages and neutrophils, primary antibodies for F4/80 (Serotec) and MPO (Dako), respectively, and secondary Alexa-coupled antibodies (Invitrogen) were used. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories). All immunefluorescence pictures were taken with a fluorescence microscope (Leica) at the same exposure and intensity settings. All histological scorings and quantifications were performed in a blinded fashion.
9
Imaging Tumor Microenvironment Immune Markers
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Frozen sections of the primary tumor and liver metastases were fixed with 4% paraformaldehyde in PBS and blocked with blocking solution (5% horse sera and 1% goat sera in PBS). Immunohistochemical staining of tissue sections was performed using antibodies against PD-L1 (BioLegend, San Diego, CA, USA), CD31 (BD Biosciences, San Jose, CA, USA), fibrinogen (Abcam, Cambridge, MA, USA), and various immune cells, such as CD8 (Invitrogen, Waltham, MA, USA), F4/80 (Abd Serotec, Raleigh, NC, USA), and CD45 (BD Pharmingen, San Diego, CA, USA). To image delivery of iv injected rat anti-PD-L1 IgG or isotype control IgG in tumors, the tissue sections were incubated with Alexa-fluor 647-labeled anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA). All images were acquired using laser scan confocal microscopy (Nikon Inc., Tokyo, Japan). ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the area fractions of positive staining on the captured fluorescence images [15 ]. All graphs are plotted based on the area fractions, and each symbol represents an individual imaging analysis.
10
Immunostaining of Paraffin Sections
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Immunostaining was performed on 3-μm paraffin sections using a microwave-based antigen retrieval technique.41 (link) The antibodies utilized in the present study included collagen I (Southern Biotech), α-SMA (Sigma) and F4/80 (Serotec).
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