Ab152

Manufactured by Abcam

Sourced in United Kingdom

AB152 is a monoclonal antibody that recognizes the human CD11c antigen. CD11c is a type I transmembrane protein and a member of the integrin family, which is expressed on the surface of dendritic cells, monocytes, and macrophages.

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18 protocols using ab152

Immunohistochemical Labeling of Goldfish Brain

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After overnight fixation, female goldfish brains were washed and embedded in Shandon Cryomatrix (Thermo Scientific), snap frozen in liquid nitrogen, and kept at −80°C. Frozen blocks were sectioned and blocked in blocking buffer (0.3% Triton PBS containing 1% bovine serum albumin (BSA) for 45 min at RT. Brain section and fixed cell slides were covered with the anti-GFAP (mouse, 1:800, Millipore, MAB360) (Forlano et al., 2001 (link)), anti-aromatase B (rabbit, 1:800, from the lab of O. Kah (Menuet et al., 2003 (link); Pellegrini et al., 2007 (link)), anti-D1R antibody (rabbit, 1:500, Acris, AP09962PU-N), or anti-TH antibody (rabbit, 1:500, Abcam, AB152) (Yamamoto et al., 2011 (link)) then incubated with donkey anti-rabbit Alexa fluor 488 (1:500, Molecular Probes) and goat anti-mouse Alexa fluor 596 (1:500, Molecular Probes) for 1 h at RT. Slides were washed and mounted with the antifading medium Vectashield with 4,6-diamino-2-phenylindole (DAPI). Images were taken by Nikon A1RsiMP confocal microscope with Nikon's Imaging Software NIS-Elements. Neuroanatomical nomenclature follows Peter and Gill (Bannerman et al., 2006 (link)).

Xing L., McDonald H., Da Fonte D.F., Gutierrez-Villagomez J.M, & Trudeau V.L. (2015). Dopamine D1 receptor activation regulates the expression of the estrogen synthesis gene aromatase B in radial glial cells. Frontiers in Neuroscience, 9, 310.

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Visualizing rAAV-Mediated Gene Expression

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Immunohistochemistry (IHC) was performed on free-floating sections through injected brain regions in order to visualize the location and spread of rAAV-mediated expression, in addition to providing an estimation of transduction efficiency. Sections were washed in 0.05M phosphate buffer containing 0.1% Triton x-100 (PB-TX) and incubated overnight in either 1:2000 rabbit antiTH (Millipore AB152), 1:1000 chicken anti-GFP (Abcam 13970) or 1:2000 goat anti-FLAG (Abcam 1257). Following overnight incubation in primary antibody, sections were washed in PB-TX and incubated in either 1:500 Cy-3 conjugated goat anti-rabbit (Jackson immune research 111-165-003), 1:1000 Alexafluor 488 donkey anti-goat (Invitrogen A11055) or 1:1000 Alexafluor 488 goat anti-chicken (Invitrogen A11039) secondary antibodies for 1h at room temperature and protected from light. Sections were then washed and covered slipped using ProLong Gold antifade reagent (Molecular Probes, Eugene, OR). Sections were viewed on a Nikon TE-2000-U-Fluorescence Microscope (Melville, NY)

Benskey M.J., Manfredsson F.P., Lookingland K.J, & Goudreau J.L. (2014). The Role of Parkin in the Differential Susceptibility of Tuberoinfundibular and Nigrostriatal Dopamine Neurons to Acute Toxicant Exposure. Neurotoxicology, 46, 1-11.

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Quantitative Immunofluorescence Analysis of Alphasynuclein

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Diaminobenzidine and fluorescence labeling was performed as described.¹⁰ Briefly, sections were blocked in 10% normal donkey serum and incubated overnight at 4°C with Abs to human αS (15G7, 1:1,000, Enzo), antiphosphorylated 129 αS (ab51253, 1:4,000, Abcam), LAMP1 (ab25245, 1:500, Abcam), LC3B (5F10, 1:500, NanoTools), perilipin (PLIN; G2 sc‐390169; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and tyrosine hydroxylase (TH; ab152, 1:500, Abcam). This was followed by incubation with fluorescein isothiocyanate–conjugated secondary Abs (1:500 in PBS) for 3 hours at room temperature. Confocal microscopy was conducted with a Zeiss (Oberkochen, Germany) LSM 710 confocal scanning laser microscope. An ImageJ plug‐in called “colocalization highlighter” created a mask of either LAMP1 or PLIN pixels that overlapped with pS129‐αS pixels. The sizes of the colocalized pixels on the resultant 8‐bit images were quantified using the analyze particle function plug‐in of ImageJ. For striatal TH evaluation, 3 images within the dorsolateral striatum were acquired per section in 3 coronal sections (bregma 0.97–0.37mm), n = 5 mice per group. Fiji color thresholding was used to calculate the respective area fraction covered by TH staining; imaging was performed using a Zeiss LSM 780 confocal scanning laser microscope (×40 field planarity apochromate (PL APO) oil immersion objective).

Nuber S., Nam A.Y., Rajsombath M.M., Cirka H., Hronowski X., Wang J., Hodgetts K., Kalinichenko L.S., Müller C.P., Lambrecht V., Winkler J., Weihofen A., Imberdis T., Dettmer U., Fanning S, & Selkoe D.J. (2020). A Stearoyl–Coenzyme A Desaturase Inhibitor Prevents Multiple Parkinson Disease Phenotypes in α‐Synuclein Mice. Annals of Neurology, 89(1), 74-90.

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Striatal Protein Expression Analysis

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Harvested tissues obtained from the striatum at 72 hour post-MA or saline exposure were lysed by sonication following their suspension in RIPA lysis buffer containing a protease inhibitor cocktail, as previously described (Filichia et al., 2016 (link)). The resulting homogenate was centrifuged at 14,000 RPM for 20 minutes in a chilled chamber, and the supernatant was removed and handled separately. Protein concentrations were determined using the Bradford absorbance assay. Equal amounts of protein were then resuspended with 5x sample buffer (with beta-mercaptoethanol), and were separated by electrophoresis on SDS-PAGE gels (Bio-Rad) and transferred onto a PVDF membrane (Bio-Rad). Thereafter, membranes were probed with primary antibodies in 1:1000 dilution (rabbit anti-tyrosine hydroxylase [Millipore AB152], rabbit anti-b-actin [Abcam ab8227]) overnight in a cold room and subsequently with HRP-labelled secondary antibodies in 1:2000 dilution (goat anti-rabbit [Jackson 111-035-003]) for 1 hour at room temperature. Protein bands were visualized using an ECL kit (Thermo Fisher) and analysis was performed using NIH ImageJ software.

Lu T., Kim P.P., Greig N.H, & Luo Y. (2017). Dopaminergic neuron-specific deletion of p53 gene attenuates methamphetamine neurotoxicity. Neurotoxicity research, 32(2), 218-230.

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Retinal Cell Proliferation Immunohistochemistry

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Cryostat sections (10 μm in thickness) of each eyeball were fixed in cold acetone, and blocked with 2% normal bovine serum for 1h at room temperature. Sections of central retina were incubated with primary antibodies for TH (Millipore, AB152, 1:1000) and Ki67 (Abcam, ab15580, 1:300) overnight at 4°C and washed thoroughly with PBS. After further incubation in FITC-conjugated IgG (Invitrogen, 1:300), sections were counterstained with DAPI (Vector, H-1200) mounted, and photographed using a confocal laser scanning microscope (Fluoview 1000, Olympus, Tokyo, Japan).

Liu Z., Qiu F., Li J., Zhu Z., Yang W., Zhou X., An J., Huang F., Wang Q., Reinach P.S., Li W., Chen W, & Liu Z. (2015). Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice. PLoS ONE, 10(10), e0141144.

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Immunohistochemical Staining of Zebrafish Brain

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Zebrafish were fixed in 4% paraformaldehyde (PFA) at 4°C for 16 hours, washed in phosphate buffered saline (PBS), and then dissected to expose the brain. The dissected zebrafish were washed in PBS + 0.3% Triton (PBST), treated with acetone for 10 minutes at −20°C, washed with PBST and then blocked overnight in PBS-T + 1% DMSO + 4% normal goat serum (NGS) at 4°C. After blocking, zebrafish were incubated in primary antibodies to TH (rabbit polyclonal, Millipore, cat # AB152; 1:1000) or GFP (chicken polyclonal, Abcam, Cat # AB13970; 1:5000) for 16 hours at 4°C. Following incubation in secondary antibody (AlexaFluor 488 goat-anti-rabbit IgG, AlexaFluor 488-goat-anti-chicken IgG, or AlexaFluor 647 goat-anti-rabbit IgG, Invitrogen; 1:1000), for 16 hours at 4°C in the dark, zebrafish were washed in PBS, and passed through a series of successively higher concentrations of glycerol to 80% for imaging.

Dukes A.A., Bai Q., Van Laar V.S., Zhou Y., Ilin V., David C.N., Agim Z.S., Bonkowsky J.L., Cannon J.R., Watkins S.C., St. Croix C.M., Burton E.A, & Berman S.B. (2016). Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure. Neurobiology of disease, 95, 238-249.

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Immunocytochemical Analysis of Terminally Differentiated Cells

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Terminally differentiated cell cultures (day 50) were fixed in 4% PFA for 15 min and then washed three times with PBS. For immunocytochemistry, the cells were blocked for 1–3 h in PBS +5% donkey serum +0.1% Triton X-100 before adding the primary antibodies solution. Primary antibodies were: rabbit anti-TH (1:1000, Merck Millipore, #AB152), anti-mouse anti-Cre (1:500 ab24607), chicken anti-GFP (1:1000, Abcam, #ab13970). After incubation with primary antibodies overnight at 4 °C, the cells were washed three times before adding fluorophore-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratories) and DAPI (1:500). The cultures were incubated with secondary antibodies for 2 h and finally washed three times.

Fiorenzano A., Birtele M., Wahlestedt J.N, & Parmar M. (2021). Evaluation of TH-Cre knock-in cell lines for detection and specific targeting of stem cell-derived dopaminergic neurons. Heliyon, 7(1), e06006.

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Immunohistochemistry of Brain Tissues

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After the completion of the behavioral experiments, mice were terminally anesthetized (euthasol 240 mg/kg, 200 µl, i.p.) and intracardially perfused with PBS then 4% paraformaldehyde (PFA). Brains were extracted and postfixed overnight (4% PFA, 4°C). Coronal slices (100 µm) were obtained by vibratome (Leica VT 1200). Sections were rinsed with 0.6% Triton X-100 in 1× PBS (PBST; 6 × 60 min) and blocked in 10% normal donkey serum (NDS) in PBST [60 min, room temperature (RT)]. Primary antibodies: chicken polyclonal GFP (ab13970 Abcam; 1:500) and rabbit polyclonal anti-tyrosine hydroxylase (TH; ab152 Abcam; 1:500), were applied in 2% NDS in PBST (48 h 4°C) before washing (6 × 60 min PBST) and secondary incubation with species specific Alexafluor IgG secondary antibodies (60 min, RT, Invitrogen; 1:500). Tissues were washed again in 0.1% PBST (6 × 60 min), then mounted using DAPI Fluoromount-G (0100–20, SouthernBiotech). Images were acquired using a 20× oil objective on an Olympus microscope (see sample images in Extended Data Fig. 2-3).

Cataldi S., Lacefield C., Shashaank N., Kumar G., Boumhaouad S, & Sulzer D. (2022). Decreased Dorsomedial Striatum Direct Pathway Neuronal Activity Is Required for Learned Motor Coordination. eNeuro, 9(5), ENEURO.0169-22.2022.

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Immunofluorescence Staining of Neuronal Markers

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Immunofluorescence assays were performed as described in Caiazzo et al. 2011 (link). Primary antibodies used TH (Millipore ab-152), TUJ1 (Abcam ab-18207, www.abcam.com/beta-III-Tubulin-antibody-ab18207.pdf), VMAT2 (Chemicon, ab1598p). Detection was performed for 40 min at room temperature with the following secondary antibodies: goat-anti-mouse immunoglobulin (Ig)G (Alexa Fluor 594; Thermo Scientific); chicken anti-rabbit IgG (Alexa Fluor 488; Thermo Scientific), dilution 1:1,000. Nuclei were stained with a dilution of 1:5,000 of 5 mg/mL DAPI solution.
A scale bar is indicated in each image, and the dimension of the scale bar is specified in the figure legend.

Della Valle F., Thimma M.P., Caiazzo M., Pulcrano S., Celii M., Adroub S.A., Liu P., Alanis-Lobato G., Broccoli V, & Orlando V. (2020). Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements. Stem Cell Reports, 14(1), 60-74.

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Whole-mount Immunofluorescent Staining of Subcutaneous Adipose Tissue

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The processing of scWAT for whole-mount immunofluorescent staining was performed as previously described (21 (link)). The primary antibodies used for immunofluorescent staining included the following: anti-GFP conjugated to Alexa Fluor 488, 1:200 (Invitrogen catalog #A21311), anti-tyrosine hydroxylase (TH), 1:500 (Millipore catalog #ab152); anti-myelin protein zero (MPZ), 1:250 (Abcam catalog #31851), anti-Synaptic Vesicle Glycoprotein 2A (SV2), 1:250 (DSHB catalog #SV2), and anti-Neurofilament M (2H3), 1:500 (DSHB catalog #2H3). The secondary antibodies for whole-mount immunofluorescent staining included the following: goat anti-rabbit IgG (H + L) highly cross adsorbed Alexa Fluor Plus 594, 1:500 (Invitrogen catalog #A32740) or Alexa Fluor 594 (Invitrogen catalog #A11005), and goat anti-mouse IgG (H + L) Alexa Fluor 488, 1:500 (Invitrogen catalog #A11001). Confocal imaging was performed on either a Leica TCS SP8 DLS or Leica Stellaris microscope with HyD detectors and white light laser. The objectives used were as follows: HC PL APO ×10/0.40 CS2, HC PL APO CS2 ×20/0.75, WD 0.62 mm, dry; HC PL APO CS2 ×40/1.30 oil, and HC PL APO CS2 ×63/1.40, WD 0.14 mm, oil. The images were processed with LASX software and pseudo-colored. A 4-kernel median noise filter was applied where the entire depot was imaged (Figure 4E).

Blaszkiewicz M., Gunsch G., Willows J.W., Gardner M.L., Sepeda J.A., Sas A.R, & Townsend K.L. (2022). Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation. Frontiers in Endocrinology, 13, 864925.

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