Rabbit anti cd68 antibody

Manufactured by Abcam

Sourced in United Kingdom, United States

Rabbit anti-CD68 antibody is a primary antibody that binds to the CD68 antigen. CD68 is a glycoprotein expressed by monocytes and macrophages. This antibody can be used for the detection and localization of CD68-positive cells in various applications.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (3 mentions) Ventana immunostainer, by Abcam (2 mentions) Rabbit anti lox 1 antibody, by Abcam (2 mentions) Histone simple stain kit, by Nichirei Biosciences (2 mentions) Ventana immunostainer, by Roche (1 mentions) Bx40 upright light microscope, by Olympus (1 mentions) Histofine simple stain kit, by Nichirei Biosciences (1 mentions) Rabbit anti lc3 antibody, by Proteintech (1 mentions) Leica rm2235, by Leica camera (1 mentions) Leica cm1850uv, by Leica camera (1 mentions) Rabbit anti p62 antibody, by Proteintech (1 mentions) B 40 upright light microscope, by Olympus (1 mentions) Vectastain elite abc rabbit igg kit, by Vector Laboratories (1 mentions) Cm1850 uv, by Leica camera (1 mentions) Rm2235, by Leica camera (1 mentions) Upright microscope, by Olympus (1 mentions) Bond polymer refine kit, by Leica Biosystems (1 mentions) Bond polymer refine kit, by Leica camera (1 mentions) Axio observer d1 microscope, by Zeiss (1 mentions) Rabbit anti cd31 antibody, by Abcam (1 mentions)

12 protocols using rabbit anti cd68 antibody

Histological Analysis of Liver Tissue

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Hematoxylin and eosin staining was performed on 5 μm sections from the paraffin-embedded tissue blocks for conventional light microscopy analysis. For CD68 staining, the liver sections were de-paraffinised and immunostained with rabbit anti-CD68 antibody (Abcam) using the automated Ventana immunostainer, according to manufacturer's recommendation. For CD3 staining, the liver sections were de-paraffinised and immunostained with rabbit monoclonal anti-CD3 antibody (Ventana Medical Systems, Inc) using the automated Ventana immunostainer, according to manufacturer's recommendation. For Sirius Red staining, liver sections were de-parrinised and stained using standard protocol.

Giles D.A., Moreno-Fernandez M.E., Stankiewicz T.E., Cappelletti M., Huppert S.S., Iwakura Y., Dong C., Shanmukhappa S.K, & Divanovic S. (2016). Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis. PLoS ONE, 11(2), e0149783.

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Histological Analysis of Heart Tissue

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Hearts were dissected free from the surrounding connective tissue, and fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into slices using a microtome (Leica RM 2235 or Leica CM1850UV; Leica, Solms, Germany). The slices were then mounted onto glass slides, and histological examinations were performed. Immunohistochemistry was performed using Histofine Simple Stain kit (Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinised with xylene and then rehydrated in a descending ethanol series. Sections were treated with 3% H₂O₂ in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).

Zhang X., Liu H., Hao Y., Xu L., Zhang T., Liu Y., Guo L., Zhu L, & Pei Z. (2018). Coenzyme Q10 protects against hyperlipidemia-induced cardiac damage in apolipoprotein E-deficient mice. Lipids in Health and Disease, 17, 279.

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Immunohistochemical Analysis of LOX-1 and CD68

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Immunohistochemistry was performed using the Histone Simple stain kits (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol concentrations. The sections were treated for 15 min with 3% H₂O₂ in methanol to inactivate endogenous peroxidases and were then incubated at room temperature for 1 h with the primary antibodies against LOX-1 (rabbit anti-LOX-1 antibody, 1:200; Abcam, England) or CD68 (rabbit anti-CD68 antibody, 1:500; Abcam, England). All sections were analyzed using an Olympus B × 40 upright light microscope (Olympus, Tokyo, Japan). For each staining, totally 3 × 7 sections (7 mice) per group were analyzed and the representative images were presented. All image analyses were done by a blinded reviewer.

Xu J., Zhu L., Liu H., Li M., Liu Y., Yang F, & Pei Z. (2018). Thymoquinone reduces cardiac damage caused by hypercholesterolemia in apolipoprotein E-deficient mice. Lipids in Health and Disease, 17, 173.

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Quantifying Renal CD68+ Macrophages

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Mouse paraffin kidney sections (3 μm) from Crb2^+/+pod-Cre^Tg/+ mice, Crb2^fl/fl mice, and Crb2^fl/flpod-Cre^Tg/+ mice at 6 months of age were incubated with proteinase K (20 μg/ml) for 20 min at room temperature (n = 5 each). Staining was performed using the VECTASTAIN Elite ABC Kit (rabbit IgG) (Vector Laboratories, Burlingame, CA, USA). The primary antibody was the rabbit anti-CD68 antibody (Abcam). The sections were incubated with 1% 3,3′-Diaminobenzidine (DAB) solution and counterstained with Mayer's Hematoxylin solution. CD68-positive cells were counted in 10 randomly selected high-power fields from each mouse.

Tanoue A., Katayama K., Ito Y., Joh K., Toda M., Yasuma T., D’Alessandro-Gabazza C.N., Kawachi H., Yan K., Ito M., Gabazza E.C., Tryggvason K, & Dohi K. (2021). Podocyte-specific Crb2 knockout mice develop focal segmental glomerulosclerosis. Scientific Reports, 11, 20556.

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Immunohistochemistry of Kidney Samples

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Kidney samples were collected and fixed in 4% paraformaldehyde. Samples embedded in paraffin were cut into slices using a microtome (Leica RM2235 or Leica CM1850UV, Solms, Germany). The slices were then mounted onto glass slides, and immunohistochemistry was performed using the Histone Simple Stain Kit (Nichirei, Tokyo, Japan) according to the manufacturer's protocol. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated with serially diluted water-ethanol solution. The sections were treated with 3% H₂O₂ in methanol to inactivate endogenous peroxidases for 15 min and incubated with primary antibodies for CD68 (rabbit anti-CD68 antibody, 1 : 500; Abcam, UK) or LOX-1 (rabbit anti-LOX-1 antibody, 1 : 250; Abcam) at room temperature for 1 h. All sections were observed with ×40 objective lenses under an upright microscope (Olympus, Tokyo, Japan).

Xu J., Pei Z., Yu M., Li X., Wang L., Lin Y., Chen X, & Liu X. (2020). Combinational Use of Antiplatelet Medication Sarpogrelate with Therapeutic Drug Rosuvastatin in Treating High-Cholesterol Diet-Induced Chronic Kidney Disease in ApoE-Deficient Mice. BioMed Research International, 2020, 1809326.

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Quantifying Tissue Pathology Markers

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All immunohistochemistry staining was performed in 5-μm paraffin sections with BondRX Stainer. Briefly, the primary antibodies including rabbit anti-mouse IgG from Bond Polymer Refine kit (Leica Biosystems, Buffalo Grove, IL, ready to use), rabbit anti-CD68 antibody (Abcam, ab125212, 1:500), and rabbit anti-Collagen1 antibody (BosterBio, Pleasanton, CA, PA2140-2, 1:1000) were used for detection of necrosis, inflammation, and fibrosis, respectively. Bond Polymer Refine kit (Leica, Cat No: DS9800) was applied as the detection system. The positive cells were identified as brown in color and nuclei were stained blue. The stained slides were scanned with Aperio ScanScope AT2 scanner. The whole digital slides were viewed and analyzed by ImageScope. The positive pixel count algorithm was selected and adjusted to cover each individual positive staining for analysis. The data was presented as positivity which was obtained from the following formula: positivity (%) = positive area (pixels)/total analyzed area (pixels) × 100%.

Iskenderian A., Liu N., Deng Q., Huang Y., Shen C., Palmieri K., Crooker R., Lundberg D., Kastrapeli N., Pescatore B., Romashko A., Dumas J., Comeau R., Norton A., Pan J., Rong H., Derakhchan K, & Ehmann D.E. (2018). Myostatin and activin blockade by engineered follistatin results in hypertrophy and improves dystrophic pathology in mdx mouse more than myostatin blockade alone. Skeletal Muscle, 8, 34.

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Immunohistochemical Analysis of Tissue Markers

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All sections were deparaffinized in xylene, hydrated in graded ethanol solutions, and immunostained with peroxidase conjugated secondary antibodies or fluorescent probes according to published protocols.[18 (link), 31 (link)] The following antibodies were used: rabbit anti-CD68 antibody (1/100, Abcam), rabbit anti-Fibronectin (FN) antibody (1/100, Sigma), rabbit anti-VEGF antibody (1/100, Santa Cruz Biotechnology), Mouse anti-Dentin Matrix Protein 1 (DMP1) antibody (1/2000, a gift from Dr. Chunlin Qin from Baylor College of Dentistry), Rabbit anti-Pigment epithelium-derived factor (PEDF) antibody (1/500, Millipore), Mouse anti-von Willebrand factor (vWF) antibody (1/100, Santa Cruz Biotechnology), rabbit anti-CD31 antibody (1/100, Abcam). Alizarin red staining to visualize calcium deposition was performed as per standard procedures. All fluorescently stained sections were imaged at the University of Illinois at Chicago Research Resource Center core imaging facility. Imaging was performed using a Zeiss LSM 710 confocal microscope equipped with Zen image analysis software and peroxidase stained sections were imaged using a Zeiss Axio-observer D1 microscope. All comparative fluorescence images were obtained using the same imaging conditions.

Huang C.C., Ravindran S., Yin Z, & George A. (2014). 3-D Self-Assembling Leucine Zipper Hydrogel With Tunable Properties For Tissue Engineering. Biomaterials, 35(20), 5316-5326.

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Immunofluorescence Imaging of Macrophage Markers

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Immunofluorescence staining was used to verify the expressions of CD68 and CD163. The tissue sections of PL6 were deparaffinized and rehydrated, followed by antigen retrieval. After being blocked for 1 h in 3% bovine serum albumin (BSA) at 37°C, the tissue sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti-CD68 antibody (1:100; Abcam, Cambridge, UK) and mouse anti-CD163 antibody (1:100; Abcam). Subsequently, sections were incubated with the secondary antibodies for 1 h at 37°C, followed by counterstaining with DAPI. The tissue sections were then observed and photographed under the inverted microscope.

Xu K., Wang R., Chen Q., Liu Y., Li X., Mao L., Wang C., Gao F., Hu L., Xie H., Wang C., Zhou G, & Guan X. (2022). Microenvironment components and spatially resolved single-cell transcriptome atlas of breast cancer metastatic axillary lymph nodes: The lymph node atlas of breast cancer metastases. Acta Biochimica et Biophysica Sinica, 54(9), 1336-1348.

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Histopathological Analysis of Liver

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Liver was collected and fixed in 10% buffered formalin solution overnight, followed by paraffin embedding. Hematoxylin and eosin staining was performed on 5μm sections from the paraffin-embedded tissue blocks for conventional light microscopy. For CD68 staining, liver sections were deparaffinized and immunostained with rabbit anti-CD68 antibody (Abcam, Cambridge, MA) using the automated Ventana immunostainer, according to manufacturer’s recommendation. The cell count was performed on scanned slides using Imagescope software (Leica Biosystems, Buffalo Grove, IL).

Kaplan J.M., Nowell M., Lahni P., Shen H., Shanmukhappa S.K, & Zingarelli B. (2016). Obesity enhances sepsis induced liver inflammation and injury in mice. Obesity (Silver Spring, Md.), 24(7), 1480-1488.

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Immunohistochemical Analysis of CD68+ Cells in Mouse Liver

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Liver tissues of mice were taken and fixed immediately in 10% buffered formalin, embedded in paraffin, and cut into 4 µm sections. After blockade of inner peroxidase, sections were incubated sequentially with the first antibody solution including rabbit anti-CD68 antibody (Abcam). After three washes in PBS (pH 7·4), the sections were then incubated in secondary goat anti-rabbit immunoglobulin (Ig)G conjugated with peroxidase labeled polymer, prior to colorization using diaminobenzidine reaction and counterstained with haematoxylin. Negative controls were established using rabbit IgG instead of the first antibodies.

Guo Y., Zhang Y., Hong K., Luo F., Gu Q., Lu N, & Bai A. (2014). AMPK Inhibition Blocks ROS-NFκB Signaling and Attenuates Endotoxemia-Induced Liver Injury. PLoS ONE, 9(1), e86881.

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