After pretreatment with citrate buffer and blocking solution (see above) for 1 h at RT, three free-floating sections per animal (n = 6 per genotype and age) were stained with the following primary antibodies over-night at 4 °C: OLIG2 (1:500, AB9610, Merck Millipore), platelet-derived growth factor receptor alpha (PDGFRα, 1:250, AF1062, R&D Systems), α-syn (1:200, 15G7, ALX-804-258, Enzo Life Sciences), phosphorylated α-syn at Ser129 (pS129-α-syn, 1:500, ab51253, Abcam), and TPPP/p25α (1:200, ab92305, Abcam). For fluorescence staining, the following secondary antibodies were incubated for 1 h at RT: Alexa Fluor 647 donkey anti-rabbit (1:1000, 711-605-152, Dianova), Alexa Fluor 568 donkey anti-goat (1:1000, A11057, Life Technologies), and Alexa Fluor 488 donkey anti-rat (1:1000, A21208, Life Technologies). Nuclei were counterstained with DAPI (1:10,000, D8417, Sigma) for 10 min. To analyze the cell density in the ROI, three Z stack images were taken in each region using the fluorescence Observer microscope (Zeiss) in conjunction with the ZEN blue software. Cell density and the area of OLIG2+ cells were quantified using the cell counter plugin and the analyze particles plugin of the ImageJ software, respectively.
Af1062
Manufactured by R&D Systems
Sourced in United States
AF1062 is a protein-based product developed by R&D Systems. It is designed for use in laboratory applications, but a detailed description of its core function is not available due to the need to maintain an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab9610, by Merck Group (4 mentions)
Ab7349, by Abcam (3 mentions)
Normal donkey serum (nds), by Merck Group (2 mentions)
Ma5 15143, by Thermo Fisher Scientific (2 mentions)
L9393, by Merck Group (2 mentions)
Adi msa 102, by Enzo Life Sciences (2 mentions)
Ab18465, by Abcam (2 mentions)
Ab16667, by Abcam (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Ab92305, by Abcam (1 mentions)
A21208, by Thermo Fisher Scientific (1 mentions)
A 11057, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Observer fluorescence microscope, by Zeiss (1 mentions)
D8417, by Merck Group (1 mentions)
Ab51253, by Abcam (1 mentions)
Alexa fluor 568 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Mouse igg blocking reagent, by Vector Laboratories (1 mentions)
27 protocols using af1062
1
Multimodal Immunostaining for Oligodendrocyte Lineage and α-Synuclein
2
Oligodendrocyte Immunohistochemistry in CNS
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The corpus callosum coronal sections and the spinal cord transverse sections were immunostained to evaluate oligodendrocytes. These sections were permeabilized with 0.3% Triton X-100, and nonspecific binding sites were blocked with serum matching the species of the secondary antibody. Where anti-mouse IgG monoclonal antibodies were used, sections were additionally incubated with mouse IgG blocking reagent (Vector Laboratories, Burlingame, CA). The sections were then stained with the following primary antibodies: rabbit anti-Olig-2 (1 : 500, AB9610, Sigma-Aldrich, St. Louis, MO, RRID:AB_570666), goat anti-platelet-derived growth factor receptor alpha (PDGFRα, 1 : 200, AF1062, R&D Systems, Minneapolis, MN, RRID:AB_2236897), mouse anti-CC1 (1 : 100, OP80, Sigma-Aldrich, RRID:AB_2057371), and rat anti-myelin basic protein (MBP, 1 : 250, ab7349, Abcam, Cambridge, UK, RRID:AB_305869). The following secondary antibodies (1 : 500, Thermo Fisher Scientific) were used: Alexa Fluor 350-conjugated donkey anti-goat, Alexa Fluor 488-conjugated donkey anti-rabbit, Alexa Fluor 594-conjugated donkey anti-mouse, Alexa Fluor 594-conjugated goat anti-rat, and Alexa Fluor 594-conjugated goat anti-rabbit. Immunostained sections were observed under a fluorescence microscope (BZ9000; Keyence, Osaka, Japan).
3
Immunohistochemistry and in situ Hybridization
4
Immunocytochemical Analysis of Oligodendrocyte Progenitors
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Differentiated OPCs were fixed in 4% formaldehyde for 10 min, washed in PBS and incubated overnight at 4°C with the primary antibodies anti-MOG (Millipore, MAB5680, 1 : 200) and anti-PDGFRα (R&D Systems, AF1062, 1 : 200) in PBS/0.5% Triton/10% normal donkey serum (Sigma-Aldrich). Cells were washed with PBS and then incubated for 2 h at room temperature with Alexa Fluor-conjugated antibodies (Invitrogen, Alexa Fluor anti-goat 488, 1 : 1000 and Alexa Fluor anti-mouse 555, 1 : 1000). Cells were then mounted with mounting medium containing DAPI (Vector, H-1200).
5
Immunostaining of Vascular Markers
6
Immunofluorescence Quantification of Corpus Callosum
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For immunofluorescence staining, 30 μm frozen brain coronal brain sections were placed in DakoCytomation (S1699, Dako) antigen retrieval solution and incubated at 85°C for 20 minutes in 2 ml tubes. Following incubation of goat anti-PDGFRα (1:100; AF1062, R&D Systems) or rabbit anti-GST-π (1:200; ADI-MSA-102, Enzo) antibodies, primary antibodies were detected with Alexa 594 donkey anti-rabbit IgG (1:500; A21207, Invitrogen) or Alexa 488 donkey anti-goat IgG (1:500; A11055, Invitrogen). Sections were counterstained with DAPI (Sigma).
For image analysis of GST-π-positive or PDGFRα-positive cells, three areas of central corpus callosum per section were captured by confocal microscope (Nikon A1R). For each area, the forebrain corpus callosum was outlined and quantified using Nikon confocal software. GST-π-positive or PDGFRα-positive cells within these areas were counted either manually (for GST-π-positive cells) or using Image-Pro 6.3 software (Media Cybernetics, Bethesda, MD, USA) (for PDGFRα-positive cells) in a blinded manner by an independent person. The total number of GST-π-positive or PDGFRα-positive cells/mm2 were calculated for each section, and data from three sections were averaged. Data are expressed as mean ± standard error.
For image analysis of GST-π-positive or PDGFRα-positive cells, three areas of central corpus callosum per section were captured by confocal microscope (Nikon A1R). For each area, the forebrain corpus callosum was outlined and quantified using Nikon confocal software. GST-π-positive or PDGFRα-positive cells within these areas were counted either manually (for GST-π-positive cells) or using Image-Pro 6.3 software (Media Cybernetics, Bethesda, MD, USA) (for PDGFRα-positive cells) in a blinded manner by an independent person. The total number of GST-π-positive or PDGFRα-positive cells/mm2 were calculated for each section, and data from three sections were averaged. Data are expressed as mean ± standard error.
7
Multiplexed Labeling of Brain Cells
8
Immunohistochemical Analysis of PDGFRs
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Frozen sections from undecalcified bones were obtained using a tape transfer system.7 Immunostaining was performed for PDGFRα (1:80, AF1062, R&D Systems, Minneapolis, MN, USA) and PDGFRβ (1:100, MA5‐15143; Thermo Fisher Scientific, Waltham, MA, USA) as previously described.19 Images were acquired on an Axioscan microscope (Carl Zeiss, Thornwood, NY, USA). Following fluorescent imaging, sections were stained with Mayer's hematoxylin (Sigma–Aldrich, St. Louis, MO, USA) or 0.02% Fast green followed by 0.1% Safranin O.
9
Lipid and Immune Cell Visualization
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Frozen sections 8 µm thick were cut from human biopsies and the midbelly of mouse muscles. Lipid was visualized using an Oil Red O staining kit (American MasterTech, #KTORO) according to manufacturer’s instructions. Immunofluorescence was performed by fixing sections in chilled 10% neutral buffered formalin, blocking with 5% BSA and incubation with primary antibodies against perilipin-1 (1:250, Sigma, #P1873), PDGFRα (1:250, Cell Signaling, #3174S), Annexin A2 (1:250, Santa Cruz, #SC-9061) and F4/80 (1:500, Serotec, #MCA497). For co-labeling anti-PDGFRα (1:100, R&D Systems, #AF1062) was used. Staining was visualized using relevant secondary antibodies conjugated to AlexaFluor 488 and/or 568 (1:500, ThermoFisher). Myofiber membranes were marked using AlexaFluor 488-conjugated wheat germ agglutinin (1:500, ThermoFisher, #W11261) and coverslips were mounted using ProLong Gold with DAPI (ThermoFisher, #P36941).
10
Immunofluorescence Staining Protocol
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Formalin-fixed paraffin sections were deparaffinized, rehydrated and immersed in PBS. Antigen retrieval was performed with citric acid (0.01 M, pH 6.0), and non-specific sites of the sections were blocked by incubation with PBS containing 10% donkey serum and 0.2% Triton X-100 for 1 h. The sections were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 0.2% Triton X-100. Immune complexes were detected by incubation for 1 h at room temperature with Alexa Fluor 488-, Alexa Fluor 594- or Alexa Fluor 647-conjugated secondary antibodies at a dilution of 1:500 in PBS. The sections were then washed three times for 5 min with PBS, exposed to DAPI (D9542, Sigma) for 5 min to stain nuclei, washed briefly with PBS, mounted with ProLong Gold antifade reagent (P36930, Invitrogen) and sealed with a cover glass (0101172 or 0101192, Marienfeld). Primary antibodies were used at dilutions of 1:500 for red fluorescent protein (600401379, Rockland, or AB8181-200, Sicgen) and Ki67 (ab16667, Abcam), 1:200 for PPARG (sc-7273, Santa Cruz Biotechnology), PDGFRA (AF1062, R&D Systems) and YAP/TAZ (8418S, Cell Signaling).
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