Mice were euthanized and lumbar spinal cords were dissected under the level of T12 (using the last rib as a marker) and fixed in 4% paraformaldehyde (PFA) in PBS overnight. The samples were incubated in 30% sucrose in PBS for 24 h and then mounted in OCT. Using a cryostat, an initial 500 µm of each spinal cord was trimmed. Then, six transverse sections (thickness of 10 µm) with an interval of 100 µm were mounted on one slide, spanning a region of about 500 µm of mouse lumbar spinal cord at the level of L1-L2. The slides were air dried at room temperature and then stained as follows. Samples were permeabilized in 0.3% TritonX-100 in PBS for 30 min, then blocked in 1X Power Block (BioGenex, Fremont, CA) for 10 min at room temperature. Samples were incubated with a goat anti-ChAT antibody (EMD Millipore, Darmstadt, Germany) in 1% BSA and 0.3% TritonX-100 in PBS for 48 h at 4 °C. Samples were incubated with Alexa Fluor 555 donkey anti-goat IgG (Life Technologies, Carlsbad, California) for 2 h at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Using a Zeiss Ax10 microscope (Plan-APOCHROMAT 20X/0.8 Ph2 lens) equipped with an Axiocam HRM camera, images were taken from ventral horn areas of all spinal cord sections. Motor neuron cell bodies were counted using ImageJ software.
Goat anti chat antibody
Manufactured by Merck Group
Sourced in Israel, United States
The Goat anti-ChAT antibody is a laboratory reagent used for the detection and quantification of choline acetyltransferase (ChAT), an enzyme responsible for the synthesis of the neurotransmitter acetylcholine. This antibody is produced in goats and specifically binds to ChAT, allowing researchers to study its expression and localization in various biological samples.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 568 donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Mouse smn antibody, by BD (2 mentions)
Alexa fluor 555 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Ax10 microscope, by Zeiss (1 mentions)
Cy3 conjugated anti goat igg, by Jackson ImmunoResearch (1 mentions)
Axiocam camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Anti synaptophysin, by Thermo Fisher Scientific (1 mentions)
Anti neurofilament, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Vector m o m immunodetection kit, by Vector Laboratories (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Alexa fluor 555 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti guinea pig igg, by Thermo Fisher Scientific (1 mentions)
7 protocols using goat anti chat antibody
1
Quantifying Lumbar Spinal Cord Motor Neurons
2
Somatotopic Mapping of Motor Neurons
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For assessment of somatotopic changes in motor neurons in the nucleus ambiguus, the brains were dissected and cryoprotected in 20% glycerin in PB and frozen on dry ice. Serial coronal frozen sections (40 µm thick) were obtained by using a sliding microtome (HM440E; Thermo scientific) and separated into 4 series56 (link). We used series 1 and 3 to avoid double-counting.
In the brain specimens from pyramidal decussation to facial nucleus, two series of sections were processed for immunofluorescence staining for anti-choline acetyltransferase (ChAT) to elucidate whether the FB and/or DY (FB/DY)-labelled neurons were motor neurons. In brief, the sections were blocked with 25% BlockAce in phosphate-buffered saline (PBS) (pH 7.4) containing 0.5% Triton-X and incubated in goat anti-ChAT antibody (1:200; Merck Millipore, Billerica, MA). Then, the sections were washed in PBS and incubated in Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, West Grove, PA). After washing, the sections were mounted, dried, and cover-slipped with DPX. Motor neurons of the nucleus ambiguus were identified based on ChAT immunoreactivity and the location in the reticular formation, and double-labelled (FB/DY and ChAT) neurons in the nucleus ambiguus were counted (Fig.5 d). As for the somatotopic changes, a distribution chart was created centering on the obex from every other section.
In the brain specimens from pyramidal decussation to facial nucleus, two series of sections were processed for immunofluorescence staining for anti-choline acetyltransferase (ChAT) to elucidate whether the FB and/or DY (FB/DY)-labelled neurons were motor neurons. In brief, the sections were blocked with 25% BlockAce in phosphate-buffered saline (PBS) (pH 7.4) containing 0.5% Triton-X and incubated in goat anti-ChAT antibody (1:200; Merck Millipore, Billerica, MA). Then, the sections were washed in PBS and incubated in Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, West Grove, PA). After washing, the sections were mounted, dried, and cover-slipped with DPX. Motor neurons of the nucleus ambiguus were identified based on ChAT immunoreactivity and the location in the reticular formation, and double-labelled (FB/DY and ChAT) neurons in the nucleus ambiguus were counted (Fig.
3
Quantifying Apoptotic Motoneurons in ALS Cell Models
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After 53 days of differentiation (i.e., 4-weeks post-plating), rabbit anti-Cleaved-Caspase 3 (CC3 (Asp175); 1/400; Cell Signaling; New England Biolabs, Ltd., Ontario, Canada; Cat. No. 9661S) was used in combination with goat anti-CHAT antibody (1/100; Millipore; Cat. No. MAB144P) to detect apoptotic MNs in cultures obtained from isogenic or ALS-patient derived cells from the CS52 or the CS29 line, in the generic, phMNENRICHED, and phMNISOLATED conditions. For quantification, images in 3 random fields were taken with a 20X objective using an Axio Observer Z1 microscope connected to an AxioCam camera using ZEN software (Zeiss) and analyzed with Image J.
4
Spinal Cord and Neuromuscular Junction Imaging
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Spinal cords were fixed with 4% (v/v) formaldehyde in phosphate-buffered saline overnight. For gem counting in motor neurons and motor-neuron counting in lumbar spinal-cord segments L1–L2, paraffin-embedded 6-mm sections were treated with citrate buffer for antigen retrieval, and incubated with goat anti-ChAT antibody (Millipore) and/or mouse SMN antibody (BD Bioscience) followed by donkey anti-goat Alexa fluor 568 (Invitrogen) and/or donkey anti-mouse Alexa fluor 488 secondary antibodies. For neuromuscular junction (NMJ) staining, mice were anaesthetized by intraperitoneal injection of Nembutal (sodium pentobarbital; 50 mg/kg) or ketamine/xylazine (100 mg/kg ketamine/10 mg xylazine) and transcardially perfused with PBS and then 4% paraformaldehyde. After 24 h post-fixing, the tibialis anterior (TA) and flexor digitorum brevis 2/3 (FDB-2/3) muscles were dissected and teased into layers 5–10 fibers thick to facilitate penetration of antibodies, including anti-neurofilament (1:2000; Chemicon) and anti-synaptophysin (1:200, Invitrogen) antibodies. Acetylcholine receptors (AChRs) were labeled with Alexa Fluor 555-conjugated α-bungarotoxin (α-BTX, Invitrogen). Confocal immunofluorescence imaging was done with an LSM710 confocal microscope (Carl Zeiss) by merging Z-stacks of multiple planes into one image.
5
Immunohistochemical Analysis of Spinal Cord
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Spinal cords were fixed with 4% (v/v) formaldehyde in phosphate-buffered saline overnight. Oligonucleotide and SMN immunohistochemistry was performed as described (Hua et al. 2010 (link)). For gem counting in motor neurons and motor neuron counting in lumbar spinal cord segments L1–L2, paraffin-embedded 6-μm sections were treated with citrate buffer for antigen retrieval (Sahashi et al. 2012 (link)) and incubated with goat anti-ChAT antibody (Millipore) and/or mouse SMN antibody (BD Bioscience) followed by donkey anti-goat Alexa fluor 568 (Invitrogen) and/or donkey anti-mouse Alexa fluor 488 secondary antibodies. For NMJ staining, after perfusing and post-fixing, the longissimus capitus was dissected and teased into layers five to 10 fibers thick; NMJ was stained as previously described (Sahashi et al. 2012 (link)). Confocal immunofluorescence imaging was performed with an LSM710 confocal microscope (Carl Zeiss) by merging Z-stacks of multiple planes into one image.
6
Immunohistochemical Analysis of Gustatory Markers
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Immunohistochemistry was performed according to a previously described method using coronal cryosections of 10 μm thick [12 (link),21 (link)]. The following primary antibodies and dilutions were used: goat anti-villin antibody (1:50; #sc-7672, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Trpm5 antibody (1:500; #ACC-045, Alomone Labs, Jerusalem, Israel), goat anti-ChAT antibody (1:100; #AP144P, Millipore, Billerica, MA), mouse anti-IP3R3 antibody (1:500; #61312, BD Biosciences, San Jose, CA) with the Vector M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA). The following appropriate secondary antibodies were used: Alexa-546-conjugated anti-goat IgG antibody, Alexa-555-conjucated anti-goat IgG antibody (both from Invitrogen, Carlsbad, CA), and biotin-conjugated goat anti-rabbit IgG antibody (Vector Laboratories) with streptoavidin-Alexa-488 fluorescence (Invitrogen). We performed antigen-retrieval pretreatments in Target Retrieval Solution, pH 9.0 (Dako, Glostrup, Denmark) for 20 min at 80°C. The sections were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories) or Fluomount-G including DAPI for nuclear staining (Southern Biotechnology, Birmingham, AL).
7
Immunohistochemical Localization of BNP and CGRP/ChAT in Spinal Cord
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Free-floating sections of the spinal cord were blocked for 1 h at room temperature with 5% BSA in PBST to block non-specific binding, then incubated for 48 h at 4°C with a mixture of rabbit polyclonal antibody against rat BNP (diluted 1:2000; Millipore) and either guinea-pig polyclonal CGRP antibody (1:200; T-5053, Peninsula Lab, San Carlos, CA, USA) or goat anti-ChAT antibody (diluted 1:500; Millipore). After three rinses with PBST, sections were incubated for 3 h at room temperature with the appropriate secondary antibodies. The secondary antibodies were Alexa Fluor 555-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-guinea pig IgG, or Alexa Fluor 488-conjugated anti-goat IgG (diluted 1:500, Thermo Fisher Scientific Inc., Waltham, MA, USA). Negative control sections were subjected to the same procedures without primary antibody. After three washes with PBST, the sections were mounted on gelatin-coated glass slides and examined using a confocal laser-scanning microscope (LSM 510 META, Carl Zeiss, Heidelberg, Germany).
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