Anti-Cdh1 (E-cadherin) antibody (Gene Tex, GTX125890, 1:200) was applied to cryosections. Antibodies against BrdU (BioRad, MCA2060, 1:200), phosphorylated HistoneH3 (upstate, 06-570, 1:100) and ZO-1 (Invitrogen, 33-9100, 1:100) were applied to cryosections pretreated at 120°C for 20 min in 10 mM citrate buffer (pH 6.0). Antibodies against PCNA (clone PC10, Sigma P8825; 1:200), Pax6 (BioLegend, PRB-278P, 1:200-1:500), AQP0 (Millipore, AB3071, 1:200-1:500), Prox1 (Millipore, AB5475, 1:200 or Gene Tex, GTX128354, 1:500) and p-Smad2 (Cell Signaling, 3101S, 1:100) were applied to paraffin sections pretreated with heat [120°C for 20 min, in 10 mM citrate buffer (pH 6.0)].
Prb 278p
Manufactured by Merck Group
Sourced in Japan, Germany
The PRB-278P is a laboratory equipment product manufactured by Merck Group. It is designed for performing specific functions in a laboratory setting. The core function of the PRB-278P is to [product description]. No further details or interpretations about the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Ab10543, by Abcam (2 mentions)
Ab11316, by Abcam (2 mentions)
Ab7751, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab97959, by Fortrea (2 mentions)
D9564, by Merck Group (2 mentions)
Prb 435p, by Merck Group (2 mentions)
Z0334, by Novus Biologicals (2 mentions)
Adi spa 223, by GeneTex (2 mentions)
Ab15766, by GeneTex (2 mentions)
Gtx62440, by Merck Group (2 mentions)
Gtx11268, by Fortrea (2 mentions)
Mab377, by Fortrea (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab62734, by Abcam (2 mentions)
Scr004, by Merck Group (2 mentions)
Scr004, by Abcam (2 mentions)
14 protocols using prb 278p
1
Immunostaining of Cryosections and Paraffin Sections
2
Antibodies for Western Blotting and Immunostaining
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The antibodies used for western blotting (for human) were as follows: GFP (abcam, ab290, 1:2,000), α-tubulin (CST, 3873s, 1:2,000), GAPDH (CST, 2118s, 1:2,000), Flag (MBL, M185, 1:2,000), Myc (MBL, M047-3, 1:5,000), HA (MBL, M180-3, 1:5,000), Phospho-JNK (CST, 9255, 1:1,000), and WDR62 (bethyl, A310-550A, 1:1,000). For mouse, they were as follows: WDR62 (abcam, 1:1,000), WDR62 antibody generated by MBL company using antigen VGQGGNQPKAGPLRAGTC, Phospho-WDR62 1053T (present from Dominic) [52 (link)], and MEKK3 (CST, 5727, 1:1,000). The antibodies used for immunostaining were Sox2 (abcam, ab97959, 1:1,000), Pax6 (Covance, PRB-278P, 1:400), Tbr2 (Millipore, ab2283, 1:1,000), β-III Tubulin/Tuj1 (abcam, ab7751, 1:1,000), γ-Tubulin (abcam, ab11316, 1:1,000), α-Tubulin (CST, 3873, 1:2,000), Phosph-Histone 3 (P-H3) (abcam, ab10543, 1:1,000), Nestin (abcam, ab6142, 1:1,000), GFP (abcam, ab13970, 1:1,000), Ki67 (abcam, ab15580, 1:1,000), BrdU (abcam, ab6326, 1:500), Phospho-JNK (abcam, ab124956, 1:1,000), activated-caspase3 (abcam, ab13847, 1:1,000). Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) (Invitrogen).
3
Immunostaining of Organoid Cultures
4
Immunofluorescence Staining of Neural Cells
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Cells were fixed in 4% paraformaldehyde (PFA) for 10 min, followed by washes in PBS at room temperature (RT). Cells were then blocked with 3% donkey serum in PBS containing 0.01% Triton X-100 for 1 hr at RT, incubated with primary antibodies overnight at 4°C, and then washed with PBS and incubated with secondary antibodies for 1 hr at RT. We used primary antibodies for GFAP (1:2,000; DAKO; Z0334), S100β (1:200; NOVUS; NB110–57478), ALDH1L1 (1:200; NeuroMab; 75–140), synapsin (1:1,000; SYSY; 106103), αB-crystallin (1:200; Enzo; ADI-SPA-223), MAP2 (1:500; GeneTex; GTX11268), Tuj1 (1:6,000; Covance; PRB-435P), NeuN (1:400; Millipore; MAB377), Pax6 (1:500; Covance; PRB-278P), Sox1 (1:500; Millipore; AB15766), Oligo2 (1:200; GeneTex; GTX62440), NG2 (1:500; Millipore; MAB5384), and NKX2.2 (1:50; DSHB; 745A5). Nuclei were stained with DAPI (1:6,000; Sigma; D9564).
5
Immunofluorescence Staining of Cell Cultures
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Cells cultured on coverslips were fixed in 4% paraformaldehyde for 10 min. Cells were then incubated in a blocking and permeabilization buffer (10% donkey serum and 0.2% Triton X-100 in PBS) for 1 hr followed by primary antibody incubation overnight at 4°C. On the next day, coverslips were incubated with corresponding fluorescent-conjugated secondary antibodies (1:1,000, Jackson ImmunoResearch) for 1 hr at room temperature. Nuclei were counter stained with Hoechst 33258. Primary antibodies used in this study were, Cre (1:500, mouse immunoglobulin G [IgG], Millipore, MAB3120), PAX6 (1:1,000, mouse IgG, DSHB, PAX6), PAX6 (1:1,000, rabbit IgG, Covance, PRB-278P), NKX2.1 (1:500, mouse IgG, Millipore, MAB5460), HOXB4 (1:50, mouse IgG, DSHB, I12), FOXA2 (1:2,000, rabbit IgG, Abcam, ab40874), and GFP (1:2000, chicken IgY, Aves, GFP-1020).
6
Immunostaining of Organoid Cultures
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Immunostaining was carried out directly in the organoid device. Organoids were fixed with PFA 2.5% for two hours, washed 3 times in PBS and permeabilized using PBS-triton 0.1% for 15 minutes, followed by 30 minutes blocking (30% Horse serum, 10% Fetal Calf Serum, 0.1% Triton X-100 in PBS). The organoids were incubated for two hours of primary anti-body diluted in blocking solution (Rabbit anti-PAX6 1:200 Covance PRB-278P, Mouse anti-NeuN 1:200 MILLIPORE mab377), washed in PBS, incubated for twenty minutes with secondary anti-body 1:200 in blocking, followed by incubation with 4',6-diamidino-2-phenylindole (DAPI) for 10 min and PBS washing. Organoid devices were imaged following the immunostaining.
7
Immunofluorescence Staining of Neural Cells
8
Cryosectioning and Immunofluorescence Staining
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For cryosections, tissues were fixed in 4% PFA, dehydrated in 30% sucrose, and frozen in TFM (tissue freezing medium). Sections (thickness: E16.5, 50 mm; E18.5, 40 mm) were used for immunofluorescence staining as described previously (Xu et al., 2014; Zhang et al., 2014) . The antibodies used for immunostaining were Sox2 (Abcam, ab97959, 1:1000), Pax6 (Covance, PRB-278P, 1:1000), Tbr2 (Millipore, ab15894, 1:1000), b-III Tubulin (Abcam, ab7751, 1:1000), g-Tubulin (Abcam, ab11316, 1:1000), Phospho-Histone 3 (P-H3) (Abcam, ab10543, 1:1000), Ki67 (Abcam, ab15580, 1:1000), BrdU (Abcam, ab6326, 1:500), Activated-caspase3 (CST, 9664s, 1:500), Dcx (CST, 4604s, 1:1000), Tbr1 (Abcam, ab31940, 1:500), Foxp2 (Abcam, ab16046, 1:500), and ZIKV serum from a patient (1:100). Control serum for ZIKV was from a healthy person (1:100), and mouse serum immunized with recombinant ZIKV envelope (E) protein was from GenBank (GEO: JN860885) (1:500, used only in Figure S1B). Nuclei were stained with DAPI (Invitrogen). Sections stained for BrdU detection were subject to 10 min 1N HCl on ice and 30 min 2N HCl at 37 C prior to blocking.
9
Epithelial Protein Extraction and Western Blotting
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Fibroblasts were removed from cultures using 1xtrypsin:EDTA prior to protein extraction from epithelial cells. Protein was extracted using RIPA buffer (Thermo Scientific) plus Halt Protease Inhibitor and EDTA (Thermo Scientific), and quantified using the Pierce BCA protein assay kit (Thermo Scientific). Western blotting with 40µg of each sample (25µg for figure 6) was performed as previously described [19] but with 3x10minute washes rather than 3x20minute washes for the following antibodies: Cytokeratin 15 (CK15) (Abcam, AB52816), PAX6 (rabbit, Covance, PRB-278P), and GAPDH (Millipore, MAB374). For Mucin 16 (MUC16) (Abcam, AB134093) the following buffer was used for blocking and primary antibody incubation: TBST (1xTBS, 0.1% Tween20) + 5% BSA. TBST was used as a dilution buffer for the secondary antibody incubation step. For p63α (Cell Signaling Technology, 4892) manufacturer's instructions were followed for blocking, washing and antibody incubation steps. Primary antibody dilutions and the predicted molecular weights of the protein 2. Antibody dilutions used for western blotting and the predicted molecular weights of the protein bands of interest from the manufacturers datasheets. Where bands observed in this study differed from the predicted size this is indicated.
10
Immunohistochemical Profiling of Neural Markers
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Immunohistochemistry was performed as described previously with slight modifications39 (link)43 (link)44 (link). Coronal sections (50 μm) were made using a cryostat, permeabilized with 0.1–0.5% Triton X-100/PBS, and incubated overnight with primary antibodies, which included anti-Tbr2 antibody (Abcam, ab23345), anti-Sox2 antibody (R&D Systems, AF2018), anti-Pax6 antibody (Covance, PRB-278P), anti-NeuN antibody (Chemicon, MAB377), anti-FOXP2 antibody (Atlas, HPA000382), anti-Ctip2 antibody (Abcam, ab18465) and anti-GFP antibodies (Nacalai tesque, Japan, 04404-26; Medical & Biological Laboratories, Japan, 598). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.
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