We used commercially available specific anti-MOP and anti-OX/42 antibodies. Microglial cells were fixed for 20 minutes in 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) and then incubated with primary antibodies (rabbit anti-MOP 1 ∶ 400, mouse anti-OX/42 1 ∶ 500; Serotec) for 2 days at 4°C. After three washes in PB, double immunofluorescence was revealed by incubation for 2 h in the appropriate fluorochrome-conjugated secondary antibody (Alexa Fluor546 donkey anti-rabbit 1 : 500, Alexa Fluor488 donkey anti-mouse 1 : 500), diluted in 5% NDS. Sections were washed with PB and then coverslipped with an Aquatex mounting medium (Merck, Darmstadt, Germany). Sections without primary antibodies were used as negative controls.
Mouse anti ox 42
Manufactured by Bio-Rad
Sourced in United Kingdom
The Mouse anti-OX/42 is a laboratory reagent used for the detection and identification of the OX-42 antigen. It is a monoclonal antibody produced in mice that specifically binds to the OX-42 antigen, which is expressed on the surface of monocytes, macrophages, and microglia cells in rodents. The primary function of this antibody is to serve as a tool for researchers in the field of immunology and cell biology to study the distribution and properties of these cell types.
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Lab products found in correlation
Aquatex mounting medium, by Merck Group (2 mentions)
4 6 diamino 2 phenylindole, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Fitc conjugated rabbit anti rat igg, by Abcam (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Mouse anti gfap, by Abcam (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
4 protocols using mouse anti ox 42
1
Immunostaining of Microglial Cells
2
Immunohistochemical Detection of Opioid Receptors
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We used commercially available specific anti-MOR, anti-KOR and anti-OX/42 antibodies. Cells were fixed for 20 minutes in 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) and incubated with primary antibodies (rabbit anti-MOR, 1∶400 [60] , rabbit anti-KOR, 1∶400 [61] (link), rabbit anti-DOR, 1∶400, [62] (link) Neuromics; mouse anti-OX/42, 1∶500, Serotec) for 2 days at 4°C. After three washes in PB, double immunofluorescence was revealed by incubation for 2 h in the appropriate fluorochrome-conjugated secondary antibody, Alexa Fluor546 donkey anti-rabbit and Alexa Fluor488 donkey anti-mouse, diluted 1∶500 in 5% NDS. Sections were then washed with PB and coverslipped with an Aquatex mounting medium (Merck, Darmstadt, Germany). Sections without primary antibodies were used as negative controls.
3
Cryostatic Section Immunofluorescence for p62 and OX42
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), and frozen on methylbutane precooled with liquid nitrogen as previously reported (Pompili et al., 2011) .
Sagittal cryostatic sections (7 μm thickness) from saline and TMT-treated rats were fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 10 min. After quenching autofluorescence with 0.05 M ammonium chloride and saturation of non-specific sites with 3% normal donkey serum (BioCell Research Laboratories, Rancho Dominguez, CA, USA) and 0.1% Triton X-100, sections were incubated overnight at 4 °C with rabbit anti-p62/ SQSTM1 (1: 500; MBL, PM045) and mouse anti-OX42 (1: 100; Serotec, Oxford, UK). After washing, the sections were incubated with a mixture of donkey Dy-light 549 antirabbit IgG (1: 400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and donkey Dy-light 488-labeled antimouse IgG (1: 200; Jackson ImmunoResearch Laboratories). Negative controls were performed substituting specific immunoglobulins with an equivalent amount of non-specific immunoglobulins and omitting primary antibodies. Slides were mounted with Vectashield mounting medium, containing 4′,6′-diamino-2-phenylindole for nuclear staining (Vector Laboratories, Burlingame, CA, USA). Examinations and photographs were made using a fluorescence microscope (Eclipse E600; Nikon Instruments SpA).
Sagittal cryostatic sections (7 μm thickness) from saline and TMT-treated rats were fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 10 min. After quenching autofluorescence with 0.05 M ammonium chloride and saturation of non-specific sites with 3% normal donkey serum (BioCell Research Laboratories, Rancho Dominguez, CA, USA) and 0.1% Triton X-100, sections were incubated overnight at 4 °C with rabbit anti-p62/ SQSTM1 (1: 500; MBL, PM045) and mouse anti-OX42 (1: 100; Serotec, Oxford, UK). After washing, the sections were incubated with a mixture of donkey Dy-light 549 antirabbit IgG (1: 400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and donkey Dy-light 488-labeled antimouse IgG (1: 200; Jackson ImmunoResearch Laboratories). Negative controls were performed substituting specific immunoglobulins with an equivalent amount of non-specific immunoglobulins and omitting primary antibodies. Slides were mounted with Vectashield mounting medium, containing 4′,6′-diamino-2-phenylindole for nuclear staining (Vector Laboratories, Burlingame, CA, USA). Examinations and photographs were made using a fluorescence microscope (Eclipse E600; Nikon Instruments SpA).
4
Double-Immunofluorescence Staining of Tissue Sections
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For double-immunofluorescence staining, tissue sections were processed as described elsewhere (Park et al., 2007) . Briefly, sections were collected in PBS and sections were incubated in 0.2% Triton X-100 for 30 min and rinsed three times with 0.5% BSA. The sections were then incubated with the indicated primary antibodies (rabbit anti-TonEBP (Miyakawa et al., 1999) , 1:1000; mouse anti-OX-42, 1:400 (Serotec, UK); mouse anti-NeuN, 1:1000 (Millipore, US); mouse anti-GFAP 1:1500 (Abcam, US)) overnight at 4 °C. After washing in PBS, the sections were incubated with secondary antibodies (FITC-conjugated rabbit anti-rat IgG, 1:1000; Texas Red-conjugated donkey anti-goat IgG, 1:1000 (Abcam, USA)) for 1 h at room temperature. Floating sections were mounted on gelatin-coated slides and dried for 1 h at room temperature. Slides were mounted by adding Vectashield medium (Vector Laboratories). Slides were imaged by using an LSM 700 confocal laser scanning microscope (Carl Zeiss, Germany).
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