Cryo-sectioned 5 μm liver tissue slides were brought to room temperature and fixed with cold acetone for 8 min and then washed in PBS containing 0.05% Tween 20 (PBS-T). Nonspecific reactions were blocked with 5% normal goat serum (ab7481, Abcam, Cambridge, MA) and 5% TruStain fcX (BioLegend, San Diego, CA) in PBS-T for 1 h and then incubated with rabbit anti-mouse F4/80 (1:200, ab111101, Abcam) and rat anti-mouse Ly-6C (1:200, ab24973, Abcam) at 4°C overnight. After washing in PBS-T, the specimens were incubated with Alexa Fluor 488 goat anti-rat IgG (H+L) (1:400, #4416, Cell Signaling, Danvers, MA) and Alexa Fluor 555 goat anti-rabbit (1:400 #4413, Cell Signaling) for 1 hour at room temperature, washed again, treated with Vector TrueVIEW Autofluorescence Quenching Reagent and then counterstained with VECTASHIELD Mounting Medium with 4’,6-diamino-2-phenylindole (DAPI) (both: Vector Labs, Burlingame, CA). The Zeiss Axio Observer Microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY) equipped with the Zen pro 2.3 software was used to visualize the immunofluorescence staining for F4/80 and Ly-6C, and nuclear localization was provided by DAPI. The negative controls were obtained by incubating sections with non-specific rat IgG or rabbit IgG as described above.
Ab111101
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab111101 is a primary antibody that can be used for Western Blotting and Immunohistochemistry applications. The antibody targets a specific protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Antibody diluent reagent solution, by Thermo Fisher Scientific (3 mentions)
Vecstain abc kit, by Vector Laboratories (3 mentions)
Dab tris tablets, by Muto Pure Chemicals (3 mentions)
Ab133357, by Abcam (3 mentions)
Ab8878, by Santa Cruz Biotechnology (3 mentions)
Sc 271430, by Santa Cruz Biotechnology (3 mentions)
Cy3 or fitc conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Ab10558, by Abcam (3 mentions)
Ab183685, by Abcam (3 mentions)
Ab5694, by Abcam (3 mentions)
Ab15323, by Abcam (2 mentions)
Ab196436, by Abcam (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Cyanine3 (cy3), by Santa Cruz Biotechnology (2 mentions)
Ab209775, by Abcam (2 mentions)
Ab4059, by Abcam (2 mentions)
Ab34710, by Abcam (2 mentions)
Ab238132, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
40 protocols using ab111101
1
Immunofluorescent Staining of Murine Liver
2
Differential Pleural Cell Analysis
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MPE fluid was treated with red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) and MPE cells were centrifuged and stained with May-Grünwald-Giemsa. Slides were then mounted with Entellan (Merck Millipore, Darmstadt, Germany) and microscopically analyzed for the differential counting of pleural cells. Pleural lavage was performed by injecting 1 mL of saline intrapleurally and recovering it after 30 s. Pleural cells were enumerated with a haemocytometer, were centrifuged, were stained with May-Grünwald-Giemsa or with anti-rabbit F4/80 antibody (ab111101; Abcam, London, UK; RRID:AB_10859466) and hematoxylin, and were microscopically analyzed for the differential counting of pleural cells.
3
Immunostaining of Liver Samples
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All immunostaining experiments used 3-µm paraffin-embedded sections of liver fixed with 4% paraformaldehyde (Wako). Immunostaining used the Vectastain ABC kit (PK-4001; Vector Laboratories, Burlingame, CA), and target proteins were detected using the avidin-biotin complex (ABC) method. The primary antibodies were anti-F4/80 (ab111101; Abcam, Tokyo, Japan), anti-inducible nitric oxide synthase (iNOS) (ab15323; Abcam), anti-CD163 (ab182422; Abcam), and anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) (bs-1278R; Bioss Inc., Woburn, MA) antibodies. A fluorescence microscope (BIOREVO BZ-9000; Keyence, Osaka, Japan) captured the images of 5 sections from each mouse (5 mice from each group) at 100× magnification, and the number of positively stained cells was calculated using the BZ analyzer II (Keyence).
4
Histological Analyses of Mouse Kidney
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Histological analyses were performed as described previously54 (link),55 (link). Briefly, mouse kidneys were fixed in 4% paraformaldehyde in PBS, incubated overnight at 4 °C, and embedded in paraffin. Sections (4 μm thick) were stained with periodic acid–Schiff (PAS) and PSR. The glomerular area was measured by tracing the outline of the glomerular tuft of at least 50 glomeruli in the cortical fields of PAS-stained specimens. Fibrotic areas were measured digitally using a fluorescence microscope (BZ‐X800; Keyence, Osaka, Japan) in the cortical fields of PSR-stained specimens. Immunohistochemistry was performed as described previously56 (link). Sections were incubated with anti‐F4/80 antibodies (1:100; ab111101; Abcam, Cambridge, MA, USA). The TUNEL assay was conducted using an in situ apoptosis detection kit (MK-500; Takara Biomedicals, Tokyo, Japan). Interstitial TUNEL-positive cells were counted in 10 randomly selected cortical fields (magnification: × 200). All measurements were blinded.
5
Immunohistochemical Analysis of Lung and Liver
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Formalin-fixed paraffin-embedded lung sections were stained with Alcian blue Elastin Van Gieson and immunostained for αSMA (α-smooth muscle actin; 1:150; Dako, M0851), von Willebrand factor (1:300; Dako, A0082), NF-κB (nuclear factor kappa-B; 1:400; Cell Signaling, D14E12), F4/80 (1:100; Abcam, ab111101), iNOS (inducible NO synthase; 1:100; Abcam, ab15323), and CD206 (1:100; R&D Systems, AF2535). As isotype control, IgG and IgG2a were used. Formalin-fixed paraffin-embedded liver sections were stained with anti-F4/80 to confirm macrophage ablation. For immunohistochemical and immunofluorescent staining, a standard protocol was followed. Histological images were visualized using a Zeiss multislide scanning microscope (Imager.Z2; Carl Zeiss, Ltd) with an Axiocam 506 color camera (Zeiss) for immunohistochemical images and MRm camera (Zeiss) for immunofluorescent images. Slides were scanned sequentially using ×20 magnification objective lens, and the analysis was performed in Zen2 blue edition (Zeiss). For all tissue macrophage quantification, positively stained cells were counted in 6 fields of view at ×200 magnification and normalized to the control group.
6
Liver Macrophage Quantification in NASH
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For staining of the liver tissue, 10% formalin-fixed tissue was sliced into 4-μm-thick sections. Immunohistochemistry for F4/80 (ab111101; rabbit monoclonal to F4/80, dilution 1/80; Abcam, Cambridge, UK) was performed as follows. The dewaxed tissues were subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min using a microwave. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide (H2O2; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in PBS for 10 min at room temperature, followed by avidin-biotin blocking. The primary antibody was applied overnight in an antibody diluent reagent solution (Thermo Fisher Scientific). The secondary antibody reaction was performed using the Vecstain ABC kit (Vector Laboratories, Burlingame, CA, USA). The sections were stained by reaction with DAB TRIS tablets (Muto Pure Chemicals, Tokyo, Japan). The number of hepatic crown-like structures (hCLS) and histological features of macrophages in the liver from NASH were counted in at least 10 fields at × 200 magnification of each F4/80-stained section and expressed as the mean number/mm2.
7
Dissecting Cell Death Pathways
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The antibodies used for immunoblotting included: mouse monoclonal antibody against GAPDH (RM2002, Beijing Ray, Beijing, China); rabbit monoclonal antibodies against HtrA2 (ab75982, Abcam, Cambridge, MA, USA), p-RIPK1 (65746, Danvers, MA, CST, USA), RIPK1 (3493, CST), p-RIPK3 (93654, CST), human p-MLKL (91689, CST), human MLKL (ab184718, Abcam), and mouse p-MLKL (ab196436, Abcam); rabbit polyclonal antibodies against RIPK3 (ab56164, Abcam) and mouse MLKL (ab172868, Abcam); and goat anti-mouse (R3001, Beijing Ray) or goat anti-rabbit (R3002, Beijing Ray) HRP-conjugated secondary antibody.
The antibodies used for IHC staining included: CD11b (ab133357, Abcam), S100a9 (73425, CST, USA), HtrA2 (ab75982, Abcam), MPO (ab9535, Abcam), and F4/80 (ab111101, Abcam).
Other reagents included: DSS (36,000–50,000 kD, MP Biomedicals, Santa Ana, CA, USA), UCF-101 (Cayman Chemical, USA), Nec-1s, BV-6, Z-VAD (Selleck, Houston, TX, USA), mouse TNF-α (R&D, Minneapolis, MN, USA), Cell Counting Kit-8 (CCK-8, MedChemExpress, Monmouth Junction, NJ, USA), FITC-dextran (4 kDa, Sigma, St. Louis, MO, USA).
The antibodies used for IHC staining included: CD11b (ab133357, Abcam), S100a9 (73425, CST, USA), HtrA2 (ab75982, Abcam), MPO (ab9535, Abcam), and F4/80 (ab111101, Abcam).
Other reagents included: DSS (36,000–50,000 kD, MP Biomedicals, Santa Ana, CA, USA), UCF-101 (Cayman Chemical, USA), Nec-1s, BV-6, Z-VAD (Selleck, Houston, TX, USA), mouse TNF-α (R&D, Minneapolis, MN, USA), Cell Counting Kit-8 (CCK-8, MedChemExpress, Monmouth Junction, NJ, USA), FITC-dextran (4 kDa, Sigma, St. Louis, MO, USA).
8
Immunohistochemical Analysis of Macrophages
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Tissues were fixed in 10% neutral buffered Formalin, paraffin embedded, and standard IHC was performed using anti mouse F4/80 (ab111101; Abcam, Cambridge, MA) at 1/100 dil. followed by incubation with goat anti rabbit IgG HRP secondary Ab (ThermoFisher 31462) at 1/3000 dil. Cy3.0 TSA Plus (Perkin Elmer, Waltham, MA) was employed for detection and the InForm software (Perkin Elmer) was utilized for quantitation of F4/80 in stained tissue sections.
9
Immunohistochemical Analysis of Spinal Cord Microglia
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Spine cord tissues were fixed in 4% paraformaldehyde and dehydrated using 30% sucrose overnight. After embedding into OCT compound (Tissue Tek), tissues were cut into 16 μm section. Sections were blocked using 5% normal goat serum and then were incubated with the diluted primary antibody specific to F4/80 (ab111101, Abcam, MA, USA), CD16/32 (Catalog # 14-0161-82, Invitrogen, Waltham, MA, USA), and Arg1 (sc-271430, Santa Cruz, USA), overnight at 4 °C. Cy3 or FITC-conjugated secondary antibody (Santa Cruz) were incubated with sections at room temperature for 1 h. For cellular immunofluorescence, cells were fixed with 4% paraformaldehyde and incubated with the primary antibody specific to F4/80 and CD11b (ab8878) and then incubated with Cy3 or FITC-conjugated secondary antibodies. DAPI (C1002, Beyotime, China) was used to stain the nucleus in tissue sections and cells before capturing images. The images were acquired using a fluorescence microscope (Nikon, Japan).
10
Histological Analyses of Liver Tissue
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Sections of formalin-fixed tissue were stained with hematoxylin and eosin (H&E) and masson for visualizing histological features. To quantify inflammatory cell infiltration liver sections were incubated with CD11b (ab133357; Abcam, Cambridge, UK) and F4/80 (ab111101; Abcam) primary antibodies at 4°C overnight, followed by incubation with an HRP-conjugated secondary antibody (ANT020; Antgen, Wuhan, China).
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