E14.5 embryonic hearts were dissected into ice cold PBS, embedded in OCT (R.A. Lamb) and snap frozen in liquid nitrogen. Cryosections (14 μm thick) were fixed in 4% PFA for 15 minutes, permeabilised in PBS + 0.1% (v/v) Triton X-100 for 15 minutes and subsequently blocked with PBS + 1% (w/v) BSA + 10% (v/v) normal goat or horse serum (Vector) for 1 hour. Sections were then incubated with the following primary antibodies diluted in PBS + 0.1% (v/v) Triton X-100 for 24 hours at 4°C: rabbit polyclonal anti-mouse Wt-1 (Calbiochem, CA1026, 1:300), goat anti-Snail (Abcam, ab53519, 1:100), rabbit anti-PHH3 (Millipore, 06–570, 1:300), anti-vimentin (Proteintech, 10366-1-AP, 1:50). Sections were then incubated with species-specific biotinylated secondary antibodies (Vector) at 1:500, for 2 hours, or FITC conjugated goat anti-rabbit secondary antibody (Sigma, F9887, 1:160) (detection of PHH3). Sections were incubated with Cy3 conjugated streptavidin (GE Healthcare, PA43001), diluted 1:3000 or 1:1000 (detection of Snail) for 30 minutes. Coverslips were mounted with Vectashield with DAPI (Vector, H-1200) and sealed with nail varnish. Slides were stored in the dark at 4°C and imaged within 48 hours.
Rabbit anti phh3
Manufactured by Merck Group
Sourced in United States
Rabbit anti-pHH3 is a laboratory reagent used for the detection of phosphorylated histone H3, a marker of cell division. It is a polyclonal antibody raised in rabbits against a synthetic peptide corresponding to the phosphorylated serine 10 residue of histone H3.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rabbit anti ki67, by Leica (2 mentions)
Normal horse or goat serum, by Vector Laboratories (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Goat anti snail, by Abcam (1 mentions)
Biotinylated species specific secondary antibodies, by Vector Laboratories (1 mentions)
Cy3 conjugated streptavidin, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Vector Laboratories (1 mentions)
Fitc conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Fluoromount g mounting medium, by Electron Microscopy Sciences (1 mentions)
Dapi no 18860, by Serva Electrophoresis (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Brdu antibody, by BD (1 mentions)
Rabbit anti caspase 3, by R&D Systems (1 mentions)
Prism 5, by GraphPad (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Fitc conjugated phalloidin, by Merck Group (1 mentions)
Mouse anti ezrin, by Merck Group (1 mentions)
7 protocols using rabbit anti phh3
1
Immunofluorescence Staining of E14.5 Mouse Hearts
2
Immunostaining of Drosophila Gut Samples
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Dissected guts were fixed in 4% PFA in 1XPBS for 45 min. After fixation the guts were washed with 1XPBS for 10 min and stained with primary antibodies, diluted in 0.5% PBT (0.5% Triton (Sigma-Aldrich) in 1XPBS) + 5% normal goat serum (Thermo Fisher Scientific, Berman, Germany). Primary antibody staining was performed at 4°C overnight on an orbital shaker. Primary antibodies used were mouse anti-Dlg1 (DSHB, 1:250), chicken anti-GFP (Abcam, 1:250), rabbit anti-pHH3 (Millipore, 1:200), and mouse anti-Pros (DSHB, 1:250). On the next day, guts were washed with 1XPBS for 10 min and incubated with secondary antibodies and DAPI (1:1000; 100 μg/mL stock solution in 0.18 M Tris pH 7.4; DAPI No. 18860, Serva, Heidelberg) for at least 3 h at RT. After washing with 1XPBS for 10 min the stained guts were mounted in Fluoromount-G Mounting Medium (Electron Microscopy Sciences). Stained posterior midguts were imaged in the R5 region using an LSM 710 confocal microscope (Carl Zeiss) with ‘Plan-Apochromat 20 × /0.8 M27′ and ‘C-Apochromat 40 × /1.20 W Corr M27′ objectives. Image resolution was set to at least 2048 × 2048 pixels. Focal planes were combined into Z-stacks and images were then processed by Fiji software. Final images were assembled using Canvas X-Pro.
3
Mapping Cell Proliferation in Brinp1 Knockout
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Heterozygous dams at embryonic day 12.5 (E12.5), day 14.5 (E14.5) and day 16.5 (E16.5) were injected with a single dose of BrdU solution (100 mg/kg). Pregnant dams were killed at day 18.5 of gestation, and embryos were harvested. Embryo brains were drop fixed in 4 % PFA for 12 h at 4 °C then cryopreserved in 20 % sucrose solution. WT and Brinp1−/− embryo brains were cryopreserved in OCT medium and sectioned to 14 μm. For immunostaining of BrdU, slides were incubated in 1:7 HCL: PBS (37 % concentrated stock) at 37 °C for 1 h. Slides were then washed in PBS and permeabilized with PBS-0.1 % Triton X-100, then blocked with 10 % NGS/PBS-Triton for 30 min. A BrdU antibody (1:100, BD) was applied at RT for 2 h and counter stained with DAPI. Sections were also stained with the following antibodies: rabbit anti-Ki67 (1:1000, Leica), rabbit anti-pHH3 (1:400, Millipore) and rabbit anti-caspase 3 (1:1000, R&D systems).
Images for all immunostaining were obtained using a Nikon C1 confocal microscope. Cell count analysis were performed blind of genotype using Imaris 7.6.3. A minimum of three representative sections per mouse were analysed by dividing cortical regions into equal bins. Repeat measures two-way ANOVAs were performed for comparisons between WT and knock-out tissue. Statistical tests were performed with GraphPad Prism 5 (GraphPad).
Images for all immunostaining were obtained using a Nikon C1 confocal microscope. Cell count analysis were performed blind of genotype using Imaris 7.6.3. A minimum of three representative sections per mouse were analysed by dividing cortical regions into equal bins. Repeat measures two-way ANOVAs were performed for comparisons between WT and knock-out tissue. Statistical tests were performed with GraphPad Prism 5 (GraphPad).
4
Embryonic Cardiac Development Analysis
5
Immunofluorescence Labeling of Cell Markers
6
Immunostaining of Drosophila wing discs
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Wing discs were fixed in 4% (w/v) paraformaldehyde in PBS at room temperature for 20 min and washed three times for 5–10 min each with PBS containing 0.1% (v/v) Triton X-100 (PBSTx). The following primary antibodies were used: rat anti-Ds (1:1,000, a gift from Helen McNeill), mouse anti-Wg (1:1000, Developmental Studies Hybridoma Bank DHSB # 4D4), mouse anti-En (1:15, DHSB # 4D9), rat anti-HA (1:1000, Roche), rat anti-E-cadherin (1:10, DHSB # Dcad2), and rabbit anti-PHH3 (1:400, Sigma # H0412). All antibodies were diluted in PBSTx and 5% (v/v) goat serum and incubated overnight at 4°C. After 5 washes in PBSTx, discs were incubated at room temperature for 90 min with the appropriate Alexa-fluor secondary antibodies (Invitrogen #’s A11035, A48255, or A48265) diluted 1:200 in PBSTx and 5% goat serum. After 3 washes in PBSTx, between 5 – 40 wing discs were mounted in 40 μL of Vectashield Plus between a 18×18 mm number 1.5 coverslip (Zeiss # 474030–9000-000) and a 24×60 mm number 1.5 coverslip (VWR # 48393–251). Mounting between two coverslips allowed the samples to be imaged from both directions. The volume of mounting media used was critical so that discs were not overly compressed by the coverslips. Compression led to fluorescent signals from the peripodial membrane to be too close in z-space to the disc proper, making it difficult to distinguish the two during image processing.
7
Immunohistochemical Analysis of Embryonic Heads
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Embryo heads were dissected at E12.5 and E14.5, fixed in 4% PFA overnight, equilibrated in 30% sucrose for 48 h, cryo-embedded in OCT, then sectioned by cryostat at 10 μm. Antigen retrieval for pHH3, CC3, was performed with 10% citrate buffer; antigen retrieval for Tbr2 was performed with antigen unmasking solution (Vector Labs, Burlingame, CA, USA). Sections were blocked with 4% normal goat serum in PBST and incubated in primary antibodies overnight at 4 °C: rabbit anti-pHH3 (Sigma #H0412, 1:500), rabbit ant-CC3 (Cell Signaling, Danvers, MA, USA #9661 S, 1:300), and rabbit anti-TBR2 (AbCam #ab31940, 1:200). Sections were incubated for 1 h with Alexafluor 488-conjugated goat anti-rabbit (Thermo #A11008, 1:500), counterstained with DAPI, and sealed with ProLong Gold (Invitrogen). Sections were imaged on the Nikon C2 703 confocal microscope (Nikon Instruments, Melville, NY, USA).
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