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Ab9535

Manufactured by Abcam
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Sourced in United Kingdom, United States

Ab9535 is a primary antibody that recognizes the Phospho-CREB (Ser133) protein. It is suitable for use in various applications, including Western blotting and immunohistochemistry.

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Lab products found in correlation

Ab5103, by Abcam (10 mentions) Ab31630, by Abcam (5 mentions) Dm2500, by Leica (3 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Ab38898, by Abcam (3 mentions) Ab125212, by Abcam (3 mentions) Bz x700, by Keyence (2 mentions) S1700, by Agilent Technologies (2 mentions) Blocking one, by Nacalai Tesque (2 mentions) Goat anti rabbit secondary antibody, by Boster Bio (2 mentions) Sc 7884, by Santa Cruz Biotechnology (2 mentions) Sc 1265 r, by Santa Cruz Biotechnology (2 mentions) 3 3 diaminobenzidine dab kit, by Boster Bio (2 mentions) Ab104139, by Abcam (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Ab68672, by Abcam (2 mentions) Ab133357, by Abcam (2 mentions) Ab28364, by Abcam (2 mentions) Ab15580, by Abcam (2 mentions) Anti p65 antibody sc 372, by Santa Cruz Biotechnology (2 mentions)

85 protocols using ab9535

1

Histological Analysis of Lung Tissues

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Histological analysis was performed on the inferior lobe of the right lung. Four‐μm‐thick serial paraffin sections were deparaffinized in xylene and dehydrated in ethanol before hematoxylin and eosin staining (for evaluation of whole tissue structure) or immunostaining.
Before immunostaining, antigen retrieval was performed using antigen retrieval solution (S1700; Dako, Tokyo, Japan) and sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were then incubated with Blocking One (Nacalai Tesque, Tokyo, Japan) for 30 minutes at room temperature to block nonspecific antibody binding. Primary antibodies against cluster of differentiation 3 (CD3; 1:300 dilution, rabbit monoclonal; Nichirei Corp., #413601, Tokyo, Japan) and myeloperoxidase (MPO; 1:300 dilution, rabbit polyclonal; ab9535; Abcam, Cambridge, United Kingdom) were detected with peroxidase conjugated appropriate secondary antibodies and a diaminobenzidine substrate. Images of the lungs were captured using a digital microscope camera (BZ‐X700; Keyence, Itasca, IL) with 4× and 20× objective lenses.
Ueha R., Nativ‐Zeltzer N., Sato T., Goto T., Nito T., Belafsky P.C, & Yamasoba T. (2018). Acute inflammatory response to contrast agent aspiration and its mechanisms in the rat lung. The Laryngoscope, 129(7), 1533-1538.
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2

Immunohistochemical Analysis of Infected Mouse Skin

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Formalin fixed, paraffin embedded tissue blocks were prepared from mouse skin 2 days post-infection as follows: Tissue samples were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and prepared for histological analysis. For immunohistochemistry, 2-micron sections were immune-labeled using routine immunoperoxidase methods. Briefly, the paraffin was removed from sections with xylenes, after which they were washed in ethanol and then rehydrated in PBS. Samples were blocked 30 min in 3% H2O2 solution (Sigma-Aldrich), blocked with 10% goat serum/TRIS-buffered saline for 15 min, incubated for 2 h at room temperature with the primary antibodies, incubated 1 h with species-specific secondary antibodies and lastly developed in 3,3′-diaminobenzidine tetra hydrochloride as a chromogen. Primary antibodies used: anti-Myeloperoxidase (rabbit 1:50 ab9535, Abcam) and anti-histone H3 (citrulline R2 + R8 + R17) (rabbit 1:50 ab5103). Secondary antibody used: Goat-anti-rabbit IgG (H&L) mouse/human ads-HRP (1:200, Southern Biotech).
Gutierrez Jauregui R., Fleige H., Bubke A., Rohde M., Weiss S, & Förster R. (2019). IL-1β Promotes Staphylococcus aureus Biofilms on Implants in vivo. Frontiers in Immunology, 10, 1082.
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3

Comprehensive Hematological and Histological Analysis

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Blood was collected from tail veins into ethylenediaminetetraacetic acid (EDTA)-coated tubes. Automated peripheral blood counts were obtained using a Vet abc machine (scil). Blood smears and bone marrow cytospins were stained with May-Grünwald Giemsa solutions (Sigma). Mouse tissues were fixed in formalin and embedded in paraffin. Blocks were cut into 5 μm sections and mounted onto glass slides prior to staining with hematoxylin and eosin. For immunohistochemistry, slides were microwaved in 10 mM citric acid (pH 6.0) to retrieve antigens. Endogenous peroxidases were quenched using hydrogen peroxide and staining was performed using anti-myeloperoxidase antibody (1:100, ab9535, Abcam) diluted in TBST for 1 h at room temperature. Signal detection was accomplished using an HRP/DAB (ABC) Detection IHC kit (Abcam, ab64264). Sections were counterstained with hematoxylin before dehydration and coverslip mounting. Slide images were captured using a Nanozoomer digital slide scanner (Hamamatsu).
Supper E., Rudat S., Iyer V., Droop A., Wong K., Spinella J.F., Thomas P., Sauvageau G., Adams D.J, & Wong C.C. (2021). Cut-like homeobox 1 (CUX1) tumor suppressor gene haploinsufficiency induces apoptosis evasion to sustain myeloid leukemia. Nature Communications, 12, 2482.
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4

Immunohistochemical Analysis of MPO, IL-1β, and IL-6

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After deparaffinization, rehydration and antigen retrieval, the sections were incubated with anti-MPO antibody (1:100; ab9535; Abcam, Cambridge, UK), anti-IL-1β antibody (1:100; SC-7884; Santa Cruz Biotechnology, CA, USA), and anti-IL-6 antibody (1:100; SC-1265-R; Santa Cruz, CA, USA) overnight at 4 °C7 (link),8 (link). After incubation with goat anti-rabbit secondary antibody (Boster, Wuhan, China), the samples were visualized with a 3,3-diaminobenzidine (DAB) kit (Boster, Wuhan, China). The mounted sections were observed and photographed under a microscope at 200 × magnification (DM2500; Leica, Solms, Germany).
Guo S., Guo L., Fang Q., Yu M., Zhang L., You C., Wang X., Liu Y, & Han C. (2021). Astaxanthin protects against early acute kidney injury in severely burned rats by inactivating the TLR4/MyD88/NF-κB axis and upregulating heme oxygenase-1. Scientific Reports, 11, 6679.
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5

Histological Analysis of Liver Necrosis

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Hematoxylin and eosin (H&E) staining was used to assess the necrosis area of the liver. Mouse liver tissues were fixed in 10% formalin, embedded in paraffin and sectioned to 5 μm slides. The slides were deparaffinized and stained by a Hematoxylin-Eosin Staining Kit (Solarbio) according to the manufacturer's protocol. For IHC staining, liver sections were subjected to deparaffinization and antigen retrieval at first, then the non-specific antibody binding was blocked by using 10% bovine serum albumin (BSA; Biosharp) at room temperature for 1 h. After incubating with primary antibodies of FGF10 (ABN44, Millipore, 1:1000 dilution), Ki-67 (12202, Cell Signaling Technology, 1:400 dilution), CD68 (sc-20060, Santa Cruz Biotechnology, 1:50 dilution), or MPO (ab9535, Abcam, 1:50 dilution) at 4 °C overnight, appropriate secondary antibodies conjugated with HRP were added and incubated at room temperature. A Metal Enhanced DAB Substrate Kit (Solarbio) was used to visualize the sections followed by hematoxylin counterstaining. All images were captured by using a Nikon ECLIPSE Ni microscope.
Li S., Zhu Z., Xue M., Pan X., Tong G., Yi X., Fan J., Li Y., Li W., Dong Y., Shen E., Gong W., Wang X., Yu Y., Maeng Y.J., Li X., Lee K.Y., Jin L, & Cong W. (2021). The protective effects of fibroblast growth factor 10 against hepatic ischemia-reperfusion injury in mice. Redox Biology, 40, 101859.
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6

Immunofluorescence Analysis of Inflammatory Markers

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Paraffin-embedded sections were incubated at 60°C for 1 hour, deparaffinized with xylene twice, then subjected to graded series of ethanol. Antigen retrieval was performed with sodium citrate buffer (pH 6.0) at boiling point for 10 minutes. Permeabilization was performed with 0.2% Triton X-100. Sections were subsequently blocked with 10% donkey serum (ab7475, Abcam, Cambridge, MA) for 50 minutes at room temperature. Sections were incubated overnight with primary antibodies listed below and then were incubated with Alexa Fluor 488–conjugated Donkey Anti-Rabbit secondary antibody (ab150073, Abcam, Cambridge, MA) at room temperature for 1 hour. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, ab104139, Abcam, Cambridge, MA).
Antibodies used in immunofluorescence analysis included: Anti-SP-D antibody (Dr. Jo Rae Wright of the Duke University Medical Center for mouse SP-D Ab), Anti-NLRP3 antibody (PA5–20838, Thermo Scientific), antibody p-IκB-α (#2859, Cell Signaling Technology (CST), Boston, MA), p-NF-κB p65 (#3033, CST, Boston, MA), Anti-Myeloperoxidase (MPO) antibody (ab9535, Abcam, Cambridge, MA) and IL-1β (ab205924, Abcam, Cambridge, MA). Slides were visualized by a Nikon Research 2000 microscope, analyzed by the Image J software (NIH) for quantitative analysis in at least five randomly selected high-power (×200) fields/slides.
Yu J., Ni L., Zhang X., Zhang J., Abdel-Razek O, & Wang G. (2019). Surfactant protein D dampens lung injury by suppressing NLRP3 inflammasome activation and NF-κB signaling in acute pancreatitis. Shock (Augusta, Ga.), 51(5), 557-568.
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7

Immunofluorescent Staining of Tissue Sections

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The tissue sections were subjected to antigen retrieval and washed three times with PBS (5 min each time). A circle was drawn around the slices using a crayon, after which 3% hydrogen peroxide solution was added to the slices and incubated at room temperature for 15 min, followed by antigen repair. The dewaxed and hydrated tissue sections were placed into sodium citrate–citric acid antigen repair buffer, boiled at high pressure for 10 min in an autoclave, subjected to antigen repair, and cooled naturally to room temperature. Antigen blockade was then performed, after which normal goat blocking serum was added to the sections and incubated at room temperature for 15 min. After the blocking serum had been removed, diluted primary antibody solution (MPO, 1:25, ab9535, Abcam; vWF 1:50, IR527, FLEX, Dako, Glostrup, Denmark) was added to the sections and incubated in a humidified chamber at 4 °C overnight. The following day, the slices were removed, diluted fluorescent secondary antibody (1:100, Abcam) was added, and the slices were incubated at room temperature for 30 min. The cell nuclei were stained with DAPI and photographed under a microscope.
Li G., Wang B., Ding X., Zhang X., Tang J, & Lin H. (2021). Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation. Experimental & Molecular Medicine, 53(7), 1180-1191.
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8

Immunolabeling of Hamster Myeloperoxidase

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Samples of hamster ALA (6 h) embedded in paraffin were labeled with primary polyclonal antibody rabbit anti-MPO (1μg/ml; ab9535, Abcam, Cambridge, UK) and incubated overnight at 4°C. Afterwards, samples were incubated with the secondary antibody goat anti- rabbitt IgG-FITC (USBiological Catalog No 1193-62G). Then, they were incubated with rabbit IgG anti-E. histolytica (manufactured in our laboratory) for 2 h at RT. Subsequently, they were incubated with a secondary antibody goat anti-rabbitt IgG-Rhodamine (USBiological Catalog No 1103-19P7) for 1 h. Finally, the slides were incubated with Sudan black 0.05% in an ethanol 70% solution for 30 min and observed with fluorescence microscopy (Nikon, Eclipse CI H550S, Japan).
Cruz-Baquero A., Cárdenas Jaramillo L.M., Gutiérrez-Meza M., Jarillo-Luna R.A., Campos-Rodríguez R., Rivera-Aguilar V., Miliar-García A, & Pacheco-Yepez J. (2017). Different behavior of myeloperoxidase in two rodent amoebic liver abscess models. PLoS ONE, 12(8), e0182480.
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9

Immunohistochemical Analysis of Myeloperoxidase and TUNEL Apoptosis

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The slices from paraffin-embedded tissues were subjected to immunohistochemical staining for myeloperoxidase (MPO). The prepared slices were washed in PBS for 10 min and then boiled in 0.01 mmol citrate buffer (pH = 6) for 10 min for antigen retrieval. After incubation with hydrogen peroxide for 10 min, 5% bovine serum albumin (BSA) was applied as the blocking solution for 20 min at room temperature. Without washing, the sections were incubated with anti-Myeloperoxidase antibody (1:100) (ab9535, Abcam, Cambridge, UK) overnight at 4 °C. After being rinsed with PBS, the sections were incubated with goat anti-rabbit secondary antibody (1:500) (ab150079, Abcam, Cambridge, UK) and then visualised using a 3, 3-diaminobenzidine (DAB) kit (AR1022, Boster, Wuhan, China). Finally, images were recorded using a microscope at 100× magnification (IX73, Olympus, Tokyo, Japan). The TUNEL staining for apoptosis operation was performed with a commercial cell death detection kit purchased from Roche Diagnostics (Indianapolis, USA) according to the manufacturer’s protocol. The stained slices were observed by microscopy (IX73, Olympus, Tokyo, Japan) and images were recorded.
Pan Y., Li Y., Gao L., Tong Z., Ye B., Liu S., Li B., Chen Y., Yang Q., Meng L., Wang Y., Liu G., Lu G., Li W, & Li J. (2017). Development of a novel model of hypertriglyceridemic acute pancreatitis in mice. Scientific Reports, 7, 40799.
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10

Immunohistochemical Analysis of Pemphigus

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Formalin-fixed paraffin-embedded blocks of tissue from patients with IgG/IgA pemphigus or conventional pemphigus were cut into 5 μm thick sections for IHC staining. These sections were stained with myeloperoxidase (MPO) (ab9535; Abcam, Cambridge, UK), C5a (ab11877; Abcam, Cambridge, UK), CD89 (ab124717; Abcam, Cambridge, UK), interleukin (IL)-8 (ab18672; Abcam, Cambridge, UK), IL-17 (ab79056; Abcam, Cambridge, UK), and matrix metalloproteinase (MMP)-9 (ab76003; Abcam, Cambridge, UK). After autoclaving for antigen retrieval, staining was performed according to the manufactures’ instructions. We used secondary antibodies conjugated to peroxidase with 3-amino-9-ethylcarbazole to detect the expression of these molecules. The sections were randomly examined and evaluated blind by a dermatologist (YTC) and a pathologist (YLC). The expression of these molecules in the epidermis was categorized from grade 0 to grade 3 based on staining intensity (Supplementary Figure S1). The number of positive infiltrating cells in the dermis was calculated by averaging the values of three randomly selected high-power fields for each specimen.
Cho Y.T., Fu K.T., Chen K.L., Chang Y.L, & Chu C.Y. (2022). Clinical, Histopathologic, and Immunohistochemical Features of Patients with IgG/IgA Pemphigus. Biomedicines, 10(5), 1197.
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