Ab49777

Manufactured by Abcam

Sourced in United Kingdom

Ab49777 is a primary antibody that recognizes a specific target protein. It is suitable for use in various applications such as Western blotting and immunohistochemistry. The product details and technical specifications are available upon request.

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Lab products found in correlation

6 protocols using ab49777

Immunohistochemical Analysis of BAP1, T Cells, and Macrophages

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4-µm serial sections from paraffin-embedded tissue were cut and used for immunostaining. Immunohistochemistry (IHC) of BAP1 was performed on 74 tumors, as described previously [24 (link)]. Tumors were scored by two independent investigators as BAP1-positive or -negative based on nuclear staining. Immunofluorescence (IF) staining for T cells and macrophages was performed on 43 tumors as described previously [29 (link), 30 (link)]. T cell types were detected by primary antibodies: anti-CD3 (ab828, rabbit polyclonal; Abcam, Cambridge, MA, United States of America) and anti-CD8 (4B11, mouse monoclonal IgG2b; Novocastra, Valkenswaard, The Netherlands). To visualize the T cells, the following secondary antibodies were used: goat-anti-rabbit IgG Alexa 546 and goat-anti-mouse IgG2b Alexa 647 (Molecular Probes, Invitrogen, Breda, The Netherlands). Counts of intratumoral CD3⁺ and CD8⁺ T cells were represented as the number of cells per square millimeter. For IF staining of CD68⁺ macrophages, we used the primary mouse anti-human macrophage CD68 antibody (clone 514H12; ab49777; Abcam, Cambridge, United Kingdom), and as secondary antibody AlexaFluor IgG2a (488) goat-anti-mouse. The amount of CD68⁺ expression was determined in pixels per square millimeter.

Gezgin G., Dogrusöz M., van Essen T.H., Kroes W.G., Luyten G.P., van der Velden P.A., Walter V., Verdijk R.M., van Hall T., van der Burg S.H, & Jager M.J. (2017). Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment. Cancer Immunology, Immunotherapy, 66(7), 903-912.

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Immunohistochemical Staining Protocol

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All resection specimens in this study were fixed in 10% buffered formalin and paraffin embedded by routinely processing. Sections were cut at a thickness of 4 μm, heated at 60°C for 30 min, then deparaffinized and hydrated through a series of xylene and alcohol baths before staining. The slides were microwaved with antigen retrieval solution (citrate buffer, pH 6.0, containing 0.3% trisodium citrate and 0.04% citric acid) for 5 min. After replenishment of this solution, the slides were microwaved again for an additional 5 min and then allowed to cool for 20 min. The sections were then rinsed in PBS (phosphate-buffered saline), and immersed in 3% H₂O₂ for 15 minutes to block the endogenous peroxidase. Thereafter, the sections were incubated with 10% BSA (bull serum albumin) at room temperature for 60 minutes to block nonspecific antibodies. Immunohistochemical staining was performed with rabbit anti-Ras antibody (Abcam, ab108602), mouse anti-CD68 antibody (Abcam, ab49777), or rabbit anti-CD34 antibody (Abcam, ab81289) respectively at room temperature for 2 h. After incubation with the corresponding secondary antibodies for 20 min, the bound complex was visualized by using the SuperPicTure polymer detection kit (No.87-8963; Invitrogen).

Li W., Liang R.R., Zhou C., Wu M.Y., Lian L., Yuan G.F., Wang M.Y., Xie X., Shou L.M., Gong F.R., Chen K., Duan W.M, & Tao M. (2015). The association between expressions of Ras and CD68 in the angiogenesis of breast cancers. Cancer Cell International, 15, 17.

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Immunohistochemical Analysis of Mouse Pancreas

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For immunohistochemical staining, we used paraffin-embedded sections (5 μm) of mouse pancreas tissue. Deparaffinized tissue sections were incubated with primary mouse anti TLR-4 antibody (ab22048, Abcam), Galectin 3 antibody (ab53082, Abcam), anti-CD68 antibody (ab49777, Abcam), or Insulin antibody (ab63820, Abcam). Staining was visualized by using the mouse-specific HRP/DAB detection IHC kit (ab64259, Abcam), and sections were counterstained with Mayer's hematoxylin. Sections were photomicrographed with a digital camera mounted on a light microscope (Olympus BX51, Japan), digitized, and analyzed. Quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/) on non-overlapping 10 fields/section. Scoring and histological analysis were performed in a blinded fashion by two independent observers. The islet area was calculated using the software, and then the immunoreactive area of the islet was calculated using a threshold on the red color channel. We did not investigate the intensity of the immunopositive staining since it is not applicable to DAB chromogen. Results are presented as a mean count of positive cells per field or percentage of islet area.

Petrovic I., Pejnovic N., Ljujic B., Pavlovic S., Miletic Kovacevic M., Jeftic I., Djukic A., Draginic N., Andjic M., Arsenijevic N., Lukic M.L, & Jovicic N. (2020). Overexpression of Galectin 3 in Pancreatic β Cells Amplifies β-Cell Apoptosis and Islet Inflammation in Type-2 Diabetes in Mice. Frontiers in Endocrinology, 11, 30.

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Immunohistochemical Profiling of Mouse Lung

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For immunohistochemical staining, paraffin-embedded sections (4 μm) of mouse lung tissue were used. Heat-mediated antigen retrieval in citrate buffer (pH = 6.0) was performed. Deparaffinized tissue-sections were incubated with primary mouse anti-CD3 (sc-20047, Santa Cruz Biotechnology), anti-CD4 (ab183685, Abcam), anti-CD68 (ab49777, Abcam), anti-TNF-α antibody (ab6671, Abcam), and anti-IL-17 (ab79056, Abcam). Staining was visualized by using Mouse Specific HRP/DAB Detection IHC Kit (ab64259, Abcam) for CD3 and CD68, and rabbit specific HRP/AEC detection IHC Kit (ab94361, Abcam) for CD4, TNF-α, and IL-17. Sections were counterstained with Mayer's hematoxylin. Sections were photomicrographed with a digital camera mounted on light microscope (Olympus BX51, Japan), digitized, and analyzed.

Gazdic M., Simovic Markovic B., Jovicic N., Misirkic-Marjanovic M., Djonov V., Jakovljevic V., Arsenijevic N., Lukic M.L, & Volarevic V. (2017). Mesenchymal Stem Cells Promote Metastasis of Lung Cancer Cells by Downregulating Systemic Antitumor Immune Response. Stem Cells International, 2017, 6294717.

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Immunohistochemical Analysis of Liver Tissue

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For immunohistochemical staining we used paraffin embedded sections (5μm) of mouse liver tissue. We performed heat mediated antigen retrieval in Citrate Buffer (pH = 6.0). Deparaffinized tissue-sections were incubated with primary mouse anti-α-SMA antibody (ab7817, Abcam, Cambridge, UK) and primary mouse anti-CD68 antibody (ab49777, Abcam). Staining was visualized by using Mouse Specific HRP/DAB Detection IHC Kit (ab64259, Abcam) and sections were counterstained with Mayer's hematoxylin. Sections were photomicrographed with a digital camera mounted on light microscope (Olympus BX51, Japan), digitized and analyzed. Analysis was performed on 10 fields of a section at 40X magnification. Results are presented as mean count of positive cells per field.

Jovicic N., Jeftic I., Jovanovic I., Radosavljevic G., Arsenijevic N., Lukic M.L, & Pejnovic N. (2015). Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding. PLoS ONE, 10(7), e0134089.

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Renal Biopsy Immunofluorescence Analysis in Lupus Nephritis

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Renal biopsies of LN patients enrolled in this study were obtained and evaluated using the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification [5 (link)]. Normal renal biopsy specimens were obtained from nephrectomy specimens of patients with suprarenal epitheliomas. In the current study, proliferative LN was defined as LN except ISN/RPS Class V which include Class III, IV, III+V, IV+V.
Frozen sections of LN renal biopsy were air dried, fixed with paraformaldehyde, and incubated overnight at 4°C with a mouse monoclonal antibody raised against the human macrophage marker, CD68 (ab49777, Abcam, Cambridge, MA) or a rabbit polyclonal antibody against the human endothelial cell marker, CD34 (14486-1-AP, Proteintech, USA) and a goat polyclonal antibody against human renalase (ab31291, Abcam, Cambridge, MA). The presence of either CD68 or CD34 and renalase was then assessed using a fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse antibody and a tetramethylrhodamine (TRITC)-conjugated secondary anti-goat antibody respectively. The nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). Fluorescent micrographs were generated using a fluorescence microscope (Nikon, Japan).

Qi C., Wang L., Zhang M., Shao X., Chang X., Fan Z., Cao Q., Mou S., Wang Q., Yan Y., Desir G, & Ni Z. (2015). Serum Renalase Levels Correlate with Disease Activity in Lupus Nephritis. PLoS ONE, 10(10), e0139627.

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