Animals were given a lethal dose of sodium pentobarbital (120 mg/kg) (MWI, 710101) and either enucleated immediately or perfused intracardially with 4% PFA. Eye were removed and fixed in PFA for 15–30 minutes. Following dissection, retinas were either kept whole or immersed in 30% sucrose overnight prior to freezing in TFM (EMS, 72592) and cryosectioning at 20 μm. Immunostaining of retinal whole-mounts and cryosections was conducted as described previously (Duan et al., 2014 (link); Krishnaswamy et al., 2015 (link)). Primary antibodies used: mouse anti-β-galactosidase (DSHB, 40-1a), rabbit anti-β-galactosidase (Duan et al., 2014 (link)), rabbit anti-calretinin (Millipore, AB5054), mouse anti-calretinin (Milipore, AB1568), goat anti-ChAT (Milipore, AB144P), goat anti-Chx10 (Santa Cruz Biotechnology, 21690), sheep anti-Chx10 (Exalpha, X118OP), mouse anti-cre (Milipore, AB3120), chicken anti-GFP (Abcam, ab13790), rabbit anti-Lhx3 (gift of Dr. Sam Pfaff) (Sharma et al., 1998 (link)), rabbit anti-mcherry (Krishnaswamy et al., 2015 (link)), rabbit anti-Nfia (Active Motif, 39397), Rabbit anti-Otx2 (Millipore, AB9566), mouse anti-PkarIIb (BD Bioscience, 610625), rabbit anti-PKC (Sigma, P4334), rabbit anti-Ppp1r17 (Atlas Antibodies, HPA047819), mouse anti-Syt2 (ZIRC, Znp-1). All secondary antibodies used were purchased from either Invitrogen or Jackson ImmunoResearch.
Ab144p
Manufactured by Santa Cruz Biotechnology
The AB144P is a laboratory instrument designed for the quantitative analysis of proteins and other biomolecules. It utilizes advanced spectrophotometric techniques to accurately measure the concentration and purity of samples. The core function of the AB144P is to provide reliable and precise data for researchers and scientists working in the field of biochemistry and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Chicken anti gfp, by Aves Labs (2 mentions)
T 4103, by Merck Group (2 mentions)
Chromotek 5f8, by Proteintech (2 mentions)
Sc 14939, by Novus Biologicals (2 mentions)
Nb74555, by Novus Biologicals (2 mentions)
Ab13790, by Abcam (1 mentions)
Ab5054, by Merck Group (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab9260, by Merck Group (1 mentions)
Sc 17320, by Merck Group (1 mentions)
Ab1550, by Merck Group (1 mentions)
5 protocols using ab144p
1
Retinal Immunohistochemistry Techniques
2
Anatomical Sectioning and Immunolabeling of Brainstem
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At the end of experiments, brainstems were stored overnight in 4% paraformaldehyde, then transferred to 1X phosphate-buffered saline (PBS). To preserve anatomical landmarks enabling the alignment of fixed and fresh tissue, a ~ 400 μm section was cut from the sagittal face of each preparation. Sections were washed in PBS with 0.1% triton X-100, blocked in 10% heat-inactivated normal donkey serum, incubated for 2 h in blocking buffer, incubated overnight in primary antibodies targeting somatostatin (1:500, Invitrogen PA5-85,759), choline acetyltransferase (1:200, Millipore AB144P), and phox2b (1:100, Santa Cruz sc-376997).
The following day, specimens were washed, transferred to secondary antibody for 2 h, and then imaged using a Nikon C2 Confocal microscope (Nikon Instruments, Melville, NY). Levels were modified and filtered for clarity using ImageJ (National Institutes of Health, Bethesda, MD).
The following day, specimens were washed, transferred to secondary antibody for 2 h, and then imaged using a Nikon C2 Confocal microscope (Nikon Instruments, Melville, NY). Levels were modified and filtered for clarity using ImageJ (National Institutes of Health, Bethesda, MD).
3
Somatostatin Neuron Immunohistochemistry
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Mice at between P42–P46 for the adult time point or P10 for the developmental time point (n = 3 or 4 for each condition) were perfused with 4% paraformaldehyde (PFA) and brains were fixed overnight in 4% PFA at 4 °C. 50-μm vibratome sections were used for all histological experiments. Every 4th section was collected for the representation of each brain and the sections were processed for immunohistochemistry in order to confirm somatostatin identity, but also to amplify weak signals that could come from low levels of TVA expression.
For the immunofluorescence, brain sections were incubated 1 h at room temperature in a blocking solution containing 3% Normal Donkey serum and 0.3% Triton X-100 in PBS and incubated overnight or 48hrs at 4°C with primary antibodies: rat anti-RFP (1:1,000; Chromotek #5f8), chicken anti-GFP (1:1,000; Aves Labs #1020), rabbit anti-somatostatin (1:3,000; Peninsula Laboratories International T-4103.0050), goat anti-ChAT (1:250; Millipore AB144P), goat anti-CTGF (1:500; Santa Cruz Biotechnology sc-14939), rabbit anti-TPH2 (1:500, Novus Biologicals NB74555). Sections were rinsed three times in PBS and incubated for 60–90 min at room temperature or overnight at 4°C with the Alexa Fluor 488-, 594- or 647-conjugated secondary antibodies (1:500; Thermo Fisher Science or Jackson ImmunoResearch).
For the immunofluorescence, brain sections were incubated 1 h at room temperature in a blocking solution containing 3% Normal Donkey serum and 0.3% Triton X-100 in PBS and incubated overnight or 48hrs at 4°C with primary antibodies: rat anti-RFP (1:1,000; Chromotek #5f8), chicken anti-GFP (1:1,000; Aves Labs #1020), rabbit anti-somatostatin (1:3,000; Peninsula Laboratories International T-4103.0050), goat anti-ChAT (1:250; Millipore AB144P), goat anti-CTGF (1:500; Santa Cruz Biotechnology sc-14939), rabbit anti-TPH2 (1:500, Novus Biologicals NB74555). Sections were rinsed three times in PBS and incubated for 60–90 min at room temperature or overnight at 4°C with the Alexa Fluor 488-, 594- or 647-conjugated secondary antibodies (1:500; Thermo Fisher Science or Jackson ImmunoResearch).
4
Somatostatin Neuron Immunohistochemistry
5
Immunohistochemical analysis of brain and eye
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Animals were transcardially perfused with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde in PBS (pH 7.4) at different postnatal stages, then brains and eyeballs were dissected out and post fixed in 4% paraformaldehyde overnight at 4 °C. After washing in PBS for 1 h, the samples were immersed in 30% sucrose in PBS until saturation, followed by cryosection at 40μm thickness. The sections were washed by PBS for 3 times and blocked with blocking buffer (10% DS, 0.1% Triton, 1 × PBS) at room temperature for 2 hours. The sections were then incubated with the primary antibody at 4 °C overnight. The antibodies used for immunostaining were activated-Caspase3 (CST, 9664 s, 1:500), AXL (Santa Cruz, SC-1096, 1:200), Calretinin (Millipore, AB1550, 1:200), ChAT (Millipore, AB144P, 1:200), Sox2 (Santa Cruze, sc-17320, 1:200), Ki67 (Millipore, AB9260, 1:200), GFAP (CST,3670 s, 1:500) and Z631 (link) (2 μg/ml). After rinsing with 0.1%PBST, secondary antibody was incubated for 2 hours at room temperature. Finally, after sections were washed with 0.1%PBST, nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen,1:500).
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