Table 1 Antibodies used in this study
From: Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells
Target protein | Antigen | Species | Dilution | Source | Antibody characterization |
|---|---|---|---|---|---|
GFP | Protein isolated from Aequorea victoria | Rabbit | 1:500 | Invitrogen | Recognizes a single band at 27 kDa on western blot of GFP transgenic mouse brain. No immunostaining seen in sections not containing GFP |
GFP | Recombinant full length protein | Chicken | 1:1,000 | Abcam ab13970 | Recognizes a single band at 27 kDa on western blot of GFP transgenic mouse brain. No immunostaining seen in sections not containing GFP |
Tbr2 | Synthetic peptide from mouse Tbr2 | Rabbit l | 1:1,000 | Abcam ab23345 | The antibody recognizes a single band at 72 kDa on a western blot of whole embryonic mouse brain (manufacturer's website). No western blot staining after neutralization of antibody with Tbr2 peptide |
Tbr1 | Synthetic peptide from mouse Tbr1 | Rabbit | 1:1,000 | Millipore AB9616 | The antibody recognizes the 68 kDa Tbr1 protein on a western blot (manufacturer's data sheet). No staining seen in Tbr1 knock-out animal (manufacturer's personal communication) |
Neurog2 | Carboxyl terminus peptide of Neurogenin 2 of human origin | Goat | 1:100 | Santa Cruz SC19233 | No western blot staining after neutralization of antibody with Neurog2 peptide |
Calretinin | Recombinant human calretinin-22k | Mouse | 1:1,500 | Swant 6B3 | This antibody specifically recognizes the 29-kDa band on immunoblots of brain extracts. The antibody staining is absent in calretinin knock-out mice (manufacturer's website) |
Calbindin | recombinant rat calbindin D-28k | Rabbit | 1:5,000 | Swant CB38 | This antibody specifically recognizes the 28-kDa band on immunoblots of brain extracts. The antibody staining is absent in calretinin knock-out mice (manufacturer's website) |
Tyrosine hydroxylase | Tyrosine hydroxylase purified from PC12 cells | Mouse | 1:250 | Millipore MAB318 | Recognizes a protein of approximately 59 to 61 kDa by western blot and does not cross-react with dopamine-hydroxylase, phenylalanine hydroxylase, tryptophan hydroxylase, or phenyl ethanolamine-N-methyl transferase on western blots (manufacturer's website). This antibody stained only cells with the typical morphology and positioning of dopaminergic periglomerular cells in the olfactory bulb |
Parvalbumin | Parvalbumin purified from carp muscles | Mouse | 1:5,000 | Swant PV235 | Specifically stains the 45Ca-binding spot of parvalbumin (MW 12,000 and IEF 4.9) in a two-dimensional 'immunoblot'. No antibody staining seen in a parvalbumin knock-out mouse (manufacturer's website). |
Pax6 | Peptide from the carboxyl terminus of the mouse Pax6 protein | Rabbit | 1:250 | Covance PRB-278P | The antibody recognizes a single band at 49 kDa on a western blot of whole embryonic mouse brain (manufacturer's personal communication) |
NeuN | Purified cell nuclei from mouse brain | Mouse | 1:500 | Millipore MAB377 | Recognizes two to three bands in the 46- to 48-kDa range and possibly another band at approximately 66 kDa. Given that NeuN is defined by the antibody, it cannot be characterized. However for further discussion see [58] |
HuC/D | HuC/HuD neuronal protein | Mouse | 1:250 | Invitrogen A-21271 | Recognizes the Elav family members HuC, HuD and Hel-N1, which are all neuronal proteins. Anti-HuC/D monoclonal 16A11 does not recognize HuR, another Elav family member that is present in all proliferating cells |
GAD65 | Affinity-purified GAD from rat brain | Mouse | 1:1,000 | Abcam AB26113 | Recognizes selectively GAD65; extensively co-localizes with VGAT in GABAergic axon terminals [28] |