Ab5054

Manufactured by Merck Group

Sourced in United States

The AB5054 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose analytical device used for various scientific applications. The core function of the AB5054 is to facilitate data collection and analysis, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.

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12 protocols using ab5054

Retinal Immunohistochemistry Techniques

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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.

Shekhar K., Lapan S.W., Whitney I.E., Tran N.M., Macosko E.Z., Kowalczyk M., Adiconis X., Levin J.Z., Nemesh J., Goldman M., McCarroll S.A., Cepko C.L., Regev A, & Sanes J.R. (2016). COMPREHENSIVE CLASSIFICATION OF RETINAL BIPOLAR NEURONS BY SINGLE-CELL TRANSCRIPTOMICS. Cell, 166(5), 1308-1323.e30.

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Quantification of Calretinin in Somatosensory Cortex

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Somatosensory cortex homogenates were collected at P8 and prepared as described previously [34 (link)]. Protein samples were run on SDS-PAGE and transferred to cellulose acetate membranes. After incubation in TBS containing 5‰ Tween 20 and 5% milk for 1 h at room temperature (RT), the membranes were incubated with primary antibody (rabbit anti-Calretinin, Millipore, AB5054, 1:2000) at 4 °C overnight. After washing in 5‰ Tween 20 in TBS for 30 min, the membranes were incubated with secondary antibody (HRP-linked anti-rabbit IgG, Cell Signaling Technology, 7074S, 1:5000) in TBS buffer for 1 h at room temperature, and immunoreactive bands were visualized with an ECL kit (Thermo Scientific). Quantitative analysis was performed with ImageJ software. The intensity of bands was normalized to the intensity of the corresponding β-tubulin band. An unpaired Student’s t-test was used to determine the significance.

Su M., Liu J., Yu B., Zhou K., Sun C., Yang M, & Zhao C. (2021). Loss of Calretinin in L5a impairs the formation of the barrel cortex leading to abnormal whisker-mediated behaviors. Molecular Brain, 14, 67.

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Validation of Calretinin Antibodies

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Pre-adsorption and Western blots evaluations of polyclonal rabbit anti-calretinin (Millipore, AB5054) and monoclonal mouse anti-calretinin (Millipore, MAB1568) antibodies have been used to determine their specificity for calretinin (Baizer and Broussard, 2010 (link); Fuentes-Santamaria et al., 2005 (link)). We further validated the specificity of both antibodies in mouse using Western blot following standard protocols. In brief, P7 mouse brain lysates were run through 10% SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad). The membrane was then incubated with primary antibody at 4°C overnight. Horseradish peroxidase conjugated secondary antibodies against either mouse or rabbit IgG (Amersham) were used at 1:5000. The immunoblot bands were visualized with chemiluminescence (ECL-SuperSignal West Pico Chemilunimescence Substrate, Pierce). The total protein loaded in each blot was visualized by staining using 2% Comassie blue solution. The rabbit polyclonal and the mouse monoclonal antibodies were used with 1:6000 and 1:2000 dilutions and each showed a single band of the expected molecular weight of 29kDa (Schwaller, 2009 (link)), indicating that they specifically recognized the appropriate protein.

Liu W, & Davis R.L. (2014). Calretinin and calbindin distribution patterns specify subpopulations of type I and type II spiral ganglion neurons in postnatal murine cochlea. The Journal of comparative neurology, 522(10), 2299-2318.

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Immunohistochemical Labeling of Cochlear Cells

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For cochlear whole-mounts and neuronal cultures, preparations were fixed and permeabilized with 100% methanol at −20°C for 6 min and rinsed three times with 0.01M PBS (pH 7.4). Before the application of antibodies, specimens were incubated with 5% normal goat serum (NGS) for 1h at RT or 4°C overnight. Specimens were incubated with primary antibodies at either 4°C overnight (peripherin, calretinin and calbindin) or at RT for 1h (β-tubulin). Monoclonal mouse anti-β-tubulin (1:200, Covance, MMS 435P) was used to label all neurons and monoclonal mouse anti-peripherin (Chemicon, MAB1527) antibody to specifically identify putative type II spiral ganglion neurons. Polyclonal rabbit anti-calretinin (Millipore, AB5054) was used at 1:200. Polyclonal rabbit anti-calbindin (Swant, CB38) and monoclonal mouse anti-calretinin (Millipore) antibodies were used at 1:100. After washing 3X with PBS the preparations were incubated with 5% NGS for 1h at RT and then with one of the following secondary antibodies at 1:100 for 1h at RT: Alexa Fluor 594 conjugated goat anti-mouse; Alexa Fluor 488 conjugated goat anti-rabbit, Alexa Fluor 350 conjugated goat anti-mouse (Invitrogen). Multi-antibody-labeling was done sequentially. At the end of all the staining steps the preparations were washed with 0.01M PBS and mounted in DABCO medium (Sigma-Aldrich).

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Comprehensive Immunofluorescence Labeling Protocol

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Immunofluorescence was performed as previously described [19 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500); anti-Calbindin (Millipore, AB1778, 1:250); anti-Foxg1 (Abcam, ab18259, 1:1000); anti-GFP (Abcam, ab13970, 1:1000); anti-L1 (Millipore, MAB5272, 1:500); anti-Pax6 (BioLegend, 901,301, 1:1000); and anti-Vglut2 (Millipore, MAB5504, 1:500). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703–545-155, 1:500), Alexa Fluor 488 donkey anti-rabbit (Life, A21206, 1:500), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500), Alexa Fluor 488 donkey anti-rat (Life, A21208, 1:500), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500) and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500).

Liu B., Zhou K., Wu X, & Zhao C. (2018). Foxg1 deletion impairs the development of the epithalamus. Molecular Brain, 11, 5.

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Immunohistochemical Analysis of Interneurons

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Mice were deeply anesthetized and perfused with cold 4% paraformaldehyde solution. The fixed brains were sliced in 50 μm coronal sections using a free-floating vibratome (Leica). Sections were double stained with a primary antibody against GFP (Aves Labs) and GAD67 (Abcam; ab97739)/TUJ (synaptic system; 302302)/SOM (Santa Cruz; sc7819)/PV (Abcam; ab11427)/CR (Millipore; AB5054)/nNOS (Millipore; AB5380)/VIP (Abcam; AB22736)/Reelin (Millipore; MAB5364). Secondary antibodies were anti-chicken Alexa 488 and anti-mouse or anti-rabbit Alexa 594 (Vector; 11039, 11005, 11012 respectively). Nuclei staining were done for 5 min as a final step (Sigma; H33342). Sections were mounted onto charged slides (Fisher Scientific, Superfrost Plus) using Fluoroshield solution (Sigma, F6937).

Casalia M.L., Howard M.A, & Baraban S.C. (2017). Persistent seizure control in epileptic mice transplanted with GABA progenitors. Annals of neurology, 82(4), 530-542.

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Immunofluorescence Markers for Neural Characterization

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For immunofluorescence, we used rabbit anti-C3G (Santa Cruz Biotechnology, H-300/sc-15359, 1:150), mouse anti-N-Cadherin (Abcam # ab98952, 1:200), mouse anti-Nestin (BD Biosciences #611658, 1:200), rabbit anti-NF medium chain (Abcam #ab64300, 1:200) or mouse anti-NF medium chain (2H3, DSHB, 1:4), rabbit anti-NF light chain (Cell Signaling #2837, 1:200), mouse anti-SMI 312 (Covance SMI-312R, 1:200), rabbit anti-Cux1 (Santa Cruz Biotechnology #sc13024, 1:150), rabbit anti-Tbr1 (Abcam #31940, 1:400), rabbit anti-Tbr2 (Abcam #23345, 1:400), rat anti-β1 integrin [very late antigen (VLA), Chemicon, #MAB1997, 1:200], rabbit anti-Calretinin (Millipore #AB5054, 1:1000), mouse anti-chondroitin sulfate (clone CS-56, Sigma #C8035, 1:100), rabbit anti-Tbr1 (Abcam #ab31940, 1:500), mouse Tau-1 (Chemicon #MAB3420; 1:500), mouse anti-MAP2 (Chemicon #AB5622; 1:1000), Hoechst 33342 (Molecular probes, 1:6000) and goat secondary antibodies labeled with Alexa 488 or 594 (Molecular Probes, 1:800). The rat 9EG7 (supernatant) antibody was provided by D. Vestweber (Max Planck Institute for Molecular Biomedicine, Münster, Germany). Images were taken on a Zeiss LSM 700 confocal microscope using the Zeiss ZEN software.

Shah B., Lutter D., Bochenek M.L., Kato K., Tsytsyura Y., Glyvuk N., Sakakibara A., Klingauf J., Adams R.H, & Püschel A.W. (2016). C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development. PLoS ONE, 11(4), e0154174.

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Histological Analysis of Dlx5 Expression

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Mice were fixed by transcardiac perfusion with 4% paraformaldehyde and brains were post-fixed by overnight immersion in 4% paraformaldehyde at 4°C. Samples were cryoprotected in 30% sucrose and frozen. Cryoprotected brains were embedded in OCT (Leica, France) and 60-μm-thick free-floating cryostat sections were prepared.
For lacZ expression analysis, adult brains were fixed by perfusion with 4% PFA with no postfixation. X-gal staining was performed as described [18 (link)]. Immunohistochemistry on tissue sections was performed on free-floating sections (60 μm) of adult Dlx5^lacZ/+ brains, incubated overnight at 4°C with a chicken anti β-D-galactosidase antibody (1:2000; Aves labs BGL-1040) combined with either mouse anti PV (1:2000, Sigma P3088), rabbit anti Calretinin (1:1000, Millipore AB5054) or rat anti Somatostatin (1:1000, Millipore MAB354). Sections were then incubated for 2 hours at room temperature in the corresponding secondary fluorescent antibodies (1:300; Jackson Immunoresearch). Pictures were acquired using a Leica SP5 confocal microscope.

de Lombares C., Heude E., Alfama G., Fontaine A., Hassouna R., Vernochet C., de Chaumont F., Olivo-Marin C., Ey E., Parnaudeau S., Tronche F., Bourgeron T., Luquet S., Levi G, & Narboux-Nême N. (2019). Dlx5 and Dlx6 expression in GABAergic neurons controls behavior, metabolism, healthy aging and lifespan. Aging (Albany NY), 11(17), 6638-6656.

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Immunostaining of Utricles for Hair Cell Markers

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Utricles were permeabilized and blocked for 2–3 h at room temperature in 0.5% PBS-Triton (PBS-T) and 10% normal donkey or goat serum (Vector Labs). Samples were incubated overnight at 4°C in primary antibodies diluted in 2% donkey/goat serum and PBS-T, followed by three rinses in PBS-T. Primary antibody labeling was detected using Alexa Fluor-conjugated secondary antibodies (1:400; Invitrogen) and/or DAPI (1:1000) also in 2% donkey/goat serum, PBS-T for 3 h. Finally, samples were washed three times with PBS and mounted in SlowFade (Life Technologies) for imaging. Images were taken using a Zeiss LSM 710 microscope at 40×/1.4 Oil DIC M27 and/or 20×/0.8 M27. The following primary antibodies and dilutions were used in this study; Ms α Calbindin2 at 1:200 (MAB1568; Millipore), Rb α Calbindin2 at 1:200 (AB5054; Millipore), Gt α Anxa4 at 1:200 (AF4146; R&D Systems), Ms α Pou4f3 at 1:200 (sc-81980; Santa Cruz Biotechnology), Rb α Myosin VIIA at 1:200 (25-6790; Proteus BioScience), Gt α Spp1 at 1:200 (AF808; R&D Systems), Gt α Ocomodulin at 1:200 (sc-7446; Santa Cruz Biotechnology), Rb α Tnc at 1:200 (AB19013; Millipore), Ms α Mapt at 1:200 (4019; Cell Signaling Technology).

McInturff S., Burns J.C, & Kelley M.W. (2018). Characterization of spatial and temporal development of Type I and Type II hair cells in the mouse utricle using new cell-type-specific markers. Biology Open, 7(11), bio038083.

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Western Blot Analysis of Projection Neuron Markers

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Western blotting was carried out to detect the levels of marker proteins for each examined projection neuron type. All the rats were killed by decapitation after being deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the spinal cords were extracted and homogenized in a RIPA buffer, to which protease inhibitors had been freshly added. The homogenate was centrifuged at 2,5000 rpm for 25 min, and the protein concentration of the homogenate was determined using the BioRad DC protein assay (BioRad Laboratories). 40μg of total protein from each sample were subjected to an SDS–PAGE (10%) and transferred to a PVDF membrane (Millipore). Membranes were blocked in 5% skim milk, and incubated with rabbit anti-Cr (1:8,000, catalog AB5054, Millipore), mouse anti-Calb (1:1,000, catalog SAB4200543, Sigma-Aldrich), mouse anti-Parv (1:3,000, catalog P3088, Sigma-Aldrich) or rabbit anti-GAPDH (1:2,000, Cell Signaling Technology) overnight at 4°C. Incubated membranes were then treated with secondary antibody conjugated with horseradwash peroxidase (1:3,000, Chemicon) for 2 hrs at 37°C.

Chen S., Yang G., Zhu Y., Liu Z., Wang W., Wei J., Li K., Wu J., Chen Z., Li Y., Mu S., OuYang L, & Lei W. (2016). A Comparative Study of Three Interneuron Types in the Rat Spinal Cord. PLoS ONE, 11(9), e0162969.

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