All surgical procedures were carried out in juvenile mice (P22–28 for viral injection, P30–40 for cholera toxin labeling) under isoflurane anesthesia. For retrograde labeling of layer 5 PNs, 100 nl of cholera toxin subunit B (CTB)-Alexa Fluor-555 or CTB-Alexa Fluor-488 (ThermoFisher) was injected via glass micropipette into the lateral pons (AP: −3.1, ML: 1.2, DV: 4.7 relative to Bregma) or dorsal striatum (AP: 0.5, ML: 1.7, DV: 1.8), respectively. To express either GCaMP6s or ArchT-GFP in specific layer 5 PN populations, 700 nl of retro-AAV-Ef1α-Cre (Addgene) was injected into either the pons or striatum. A second 700 nl injection of AAV5-hSyn-Flex-GCaMP6s (Addgene) or AAV5-Cag-Flex-ArchT-GFP (UNC Vector Core) was made into V1 (AP: −3.5, ML: 2.5, DV: 0.45).
Ctb alexa fluor 555
Manufactured by Thermo Fisher Scientific
CTB)-Alexa Fluor-555 is a fluorescent dye conjugate composed of cholera toxin subunit B (CTB) and Alexa Fluor-555. It is a reagent used in biological research for labeling and tracking cell surface receptors.
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Lab products found in correlation
4 protocols using ctb alexa fluor 555
1
Retrograde Labeling and Optogenetic Manipulation of Layer 5 Neurons
2
Immunofluorescence Staining of Autoantibodies
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ANA (Hep-2) slides (AN-1012, MBL International) and anti-neutrophil cytoplasmic antibody (ANCA) slides (29419, Biorad) were removed from pouches and allowed to equilibrate at room temperature. Sera were diluted (1:20) in PBS, and 30uL was added to each well for 1h at room temperature. Slides were washed twice with blocking buffer (0.1% Tween20, 0.5% BSA in PBS). Secondary antibody goat anti-mouse IgG2c-AF488 (1079–30, Southern Biotech) and (CTB)- AlexaFluor555 (C34776, Thermo Fisher Scientific) were diluted in blocking buffer, added to the slides, and incubated for 1h at room temperature. Slides were washed twice with blocking buffer and mounted with DAPI Fluoromount-G mounting medium (0100–20, Southern Biotech).
3
Retrograde Tracing of CEl and PAG Neurons
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CTB-Alexa Fluor 555 (Invitrogen) was delivered by stereotaxic injection. Animals were sacrificed 1 week after injection and brains processed for IHC as described above. Co-localization of CTB back-labelled neurons with either PKCδ (CEl) or TH (PAG) labelling was scored by an observer blind to the experimental condition.
4
Retrograde Tracing of CEl and PAG Neurons
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