Paraffin–embedded sciatic nerve sections were rehydrated, boiled in 10 mM sodium citrate buffer (pH9) for 30 min in a microwave and subsequently treated with 0,5% Triton X–100 for 10 min. Sections were incubated for 2 h in 0.2% gelatine and 2% goat serum diluted in PBS. They were submersed in the primary antibody solution overnight at 4°C. Antibodies used: anti-neurofilament (NF; 1:200; BioLegend; SMI312), anti-myelin protein zero (MPZ; 1:200, Abcam, ab39375) and anti-myelin basic protein (MBP; 1:500; Millipore; MAB384). After vigorous washing, the sections were incubated at room temperature for 2 h with secondary antibody solution (Alexa488- and Alexa546-conjugated goat anti-mouse and -rabbit antibodies, 1:500 in PBS, Invitrogen) and counterstained using DAPI (1μg/ml PBS, 10 min).
Mab384
Manufactured by Merck Group
Sourced in United States
MAB384 is a laboratory equipment product manufactured by Merck Group. It is a versatile instrument designed for a range of research applications. The core function of MAB384 is to provide reliable and precise measurements, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Smi312, by Abcam (1 mentions)
Ab18723, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Ab1511, by Merck Group (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Mab1195, by R&D Systems (1 mentions)
Mab377, by Merck Group (1 mentions)
P3088, by Merck Group (1 mentions)
M1406, by Merck Group (1 mentions)
G3893, by Merck Group (1 mentions)
Ab13970, by Abcam (1 mentions)
Anti vglut2, by Synaptic Systems (1 mentions)
Cm3050, by Leica (1 mentions)
Anti vglut1, by Synaptic Systems (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
Lasaf 2, by Leica (1 mentions)
Tespa, by Merck Group (1 mentions)
4 protocols using mab384
1
Immunohistochemistry of Peripheral Nerve
2
Immunostaining of Neural Cell Markers
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Primary antibodies used included rabbit anti GFAP (1:1000; Z 0334; Dako, Glostrup, Denmark), mouse anti GFAP (1:1000; G3893; Sigma Aldrich, Saint Louis, USA), mouse anti MAP2 (1:500; M-1406; Sigma Aldrich, Saint Louis, USA), mouse anti-MBP (1:500; MAB 384; Millipore, California, USA), rabbit anti Iba1 (1:1000; 019–19741; Wako Pure Chemical Industries, Osaka, Japan), chicken anti GFP (1:1000; Ab13970; Abcam, Cambridge, UK), mouse anti βIII-tubulin (1:500; MAB1195; R&D Systems, Minneapolis, USA), goat anti Fgfr4 (1:500; AF2265; R&D, Minneapolis, USA), rabbit anti GAD 65/67 (1:500; AB1511; Millipore, California, USA), guinea pig anti Tlx3/Rnx (1:500; kindly provided by Thomas Müller and Carmen Birchmeier, Max-Delbrück-Center, Berlin, Germany), mouse anti NeuN (1:500; MAB377; Millipore, California, USA), rabbit anti doublecortin (1:500; ab18723; Abcam; Cambridge, UK), rat anti BrdU (1:500; Ab6326; Abcam; Cambridge, UK) and mouse anti parvalbumine (1:500; P3088, Sigma Aldrich, Saint Louis, USA).
3
Immunohistochemical Analysis of Synaptic Markers
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Mice were transcardially perfused with 4% PFA (Sigma, cat #441244). Brains were sectioned with a cryostat (Leica CM3050 S) and incubated in the following primary antibodies: anti-VGlut1 (Synaptic Systems cat#135 303, 1:1000), anti-Vglut2 (Synaptic Systems cat#135 404, 1:2000), anti-Myelin Basic Protein (Millipore MAB384, 1:150). Sections were counterstained with DAPI (300 nM, Invitrogen D3571).
Images were acquired with a laser scanning confocal microscope (Zeiss 710) using a ×63 objective (1.4 NA, VGlut) or ×20 objective (0.8 NA, MBP). Quantitative analyses were performed on a minimum of 4-6 sections per mouse, in 3-6 mice per genotype. Analysis was performed with ImageJ software.
Images were acquired with a laser scanning confocal microscope (Zeiss 710) using a ×63 objective (1.4 NA, VGlut) or ×20 objective (0.8 NA, MBP). Quantitative analyses were performed on a minimum of 4-6 sections per mouse, in 3-6 mice per genotype. Analysis was performed with ImageJ software.
4
Immunofluorescence Analysis of Peripheral Nerve Fibers
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Immunofluorescence experiments were performed as described (Della-Flora Nunes et al., 2020) . For teasing, sciatic or tibial nerves were dissected, fixed in 4% PFA for 30 min, washed with PBS and teased in slides coated with (3-Aminopropyl)triethoxysilane (TESPA; Sigma). Coating with TESPA was achieved by subsequently submerging glass slides in acetone for 1 min, 4% TESPA in acetone for 2 min and two times in acetone for 30 sec each. The teasing procedure consisted of placing a small portion of the nerve in a PBS droplet over the TESPA-coated slide, followed by careful mechanical separation of individual fibers, first using insulin syringes (0.3 ml 31 G x 8 mm) and then using modified insulin syringes containing insect pins (Fine science tools #26002-10) attached to their needle. The following primary antibodies were used: rabbit anti-JUN 1:200 (Cell signaling #9165), rabbit anti-p-S6 1:200 (Cell signaling #4858), chicken anti-P0 1:300 (Aves #PZO0308) and mouse ant-MBP 1:300 (Millipore #MAB384). Images from teased fibers were acquired at 40X magnification and 1.5X zoom using a confocal microscope Leica SP5II running the LAS AF 2.7.9723.3 software (Leica). Quantifications were performed using ImageJ Fiji v1.52p (Rueden et al., 2017 , Schindelin et al., 2012) . Four to five fields per animal were analyzed.
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