Ab13970, by BD - Product details

1

Immunostaining of Mouse Embryo Sections

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Embryos were fixed in 4% PFA at 4° C overnight followed by PBS washes. Subsequently, embryos were transferred through a 15% and 30% sucrose gradation followed by embedding in Tissue-Tek O.C.T. compound (Thermo scientific, Waltham, MA USA). 10 μM sections were prepared using Leica cryostat. For immune-staining sections were fixed in 4% PFA for 5 min on ice, washed in a 1X PBS, blocked in serum containing 3% goat serum, 3% bovine serum albumin and 0.1% Triton X-100 followed by incubation with primary antibodies overnight at 4° C. Primary antibodies used were GFP (1:200, ab13970), Runx1 (1:200, ab92336), CD41 (1:300, ab33661), CD31 (1:100, 553370, BD Pharmingen), Vegfr2/Flk1 (1:100, sc48161) and VE-cadherin (1:100, sc-6458). Secondary antibodies, anti-rabbit biotin, anti-rat biotin, anti-goat biotin and anti-chicken Alexa Fluor-488 (Life Technology) were used at 1:200 concentrations. For biotinylated antibodies signal was amplified using ABC (Vectastatin) and cy3-Tyramide amplification kit (Perkin Elmer). Images were taken using a Zeiss LSM 700 Upright confocal microscope.

Zamir L., Singh R., Nathan E., Patrick R., Yifa O., Yahalom-Ronen Y., Arraf A.A., Schultheiss T.M., Suo S., Han J.D., Peng G., Jing N., Wang Y., Palpant N., Tam P.P., Harvey R.P, & Tzahor E. (2017). Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta. eLife, 6, e20994.

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2

Whole-mount Immunostaining of Telogen Skin

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Telogen back skin was fixed in 4% PFA overnight at 4°C. After washing 10× with PBS containing 0.3% Triton X-100 (0.3% PBST) every 30 min, the skin pieces (5 mm × 1 cm) were incubated with primary antibodies in blocking buffer (0.3% PBST containing 5% goat serum and 20% DMSO) for 5 d at RT. Samples were washed with 0.3% PBST every 30 min for 10× and were incubated overnight with secondary antibodies for 3 days at RT. After washing, samples were dehydrated sequentially in 25%, 50%, and 75% methanol for 5 min and 100% methanol for 20 min, 3 times. After incubating with benzyl benzoate and benzyl alcohol (BBBA) at 2:1 (v:v) overnight at RT, samples became transparent and could be observed under the confocal microscope.
Primary antibodies were used at the following dilutions: rabbit anti-K14 (1:1000, BioLegend #905301), chick anti-GFP (1:500, Abcam #ab13970) and rat anti-CD31 (1:100, BD Biosciences #550274, clone #MEC 13.3). All secondary antibodies (TxR, FITC or Alexa-594, Jackson ImmunoResearch) were used at 1:500 dilution.

Sada A., Jacob F., Leung E., Wang S., White B.S., Shalloway D, & Tumbar T. (2016). Defining the cellular lineage hierarchy in the inter-follicular epidermis of adult skin. Nature cell biology, 18(6), 619-631.

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3

Whole-mount Immunostaining of Telogen Skin

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Telogen back skin was fixed in 4% PFA overnight at 4°C. After washing 10× with PBS containing 0.3% Triton X-100 (0.3% PBST) every 30 min, the skin pieces (5 mm × 1 cm) were incubated with primary antibodies in blocking buffer (0.3% PBST containing 5% goat serum and 20% DMSO) for 5 d at RT. Samples were washed with 0.3% PBST every 30 min for 10× and were incubated overnight with secondary antibodies for 3 days at RT. After washing, samples were dehydrated sequentially in 25%, 50%, and 75% methanol for 5 min and 100% methanol for 20 min, 3 times. After incubating with benzyl benzoate and benzyl alcohol (BBBA) at 2:1 (v:v) overnight at RT, samples became transparent and could be observed under the confocal microscope.
Primary antibodies were used at the following dilutions: rabbit anti-K14 (1:1000, BioLegend #905301), chick anti-GFP (1:500, Abcam #ab13970) and rat anti-CD31 (1:100, BD Biosciences #550274, clone #MEC 13.3). All secondary antibodies (TxR, FITC or Alexa-594, Jackson ImmunoResearch) were used at 1:500 dilution.

Sada A., Jacob F., Leung E., Wang S., White B.S., Shalloway D, & Tumbar T. (2016). Defining the cellular lineage hierarchy in the inter-follicular epidermis of adult skin. Nature cell biology, 18(6), 619-631.

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4

Multicolor Immunofluorescence Imaging Protocol

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Tissue was fixed in 4% PFA/PBS at room temperature for 10 minutes and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at −80°C. Sections were cut at 10 µm using a Leica CM3050S cryostat and incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-TH (Abcam, ab112), chicken anti-GFP (Abcam, ab13970), rat anti-CD31 (BD Pharmingen, 553370), chicken anti-β-galactosidase (Abcam, ab9361), and mouse anti-smooth muscle actin (Sigma, A5228) used at 1:500. Mouse anti-smooth muscle actin antibody was directly conjugated to Cy5 NHS ester (GE Healthcare), and unbound dye was removed on a P-30 gel exclusion column (BioRad)³⁵. Incubation with secondary antibodies was 45 minutes at room temperature. Secondary antibodies were conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or DyLight 488 (Jackson ImmunoResearch). Staining with 4’,6-diamidino-2-phenylindole (DAPI; 1 ng/ml, Life Technologies) was performed after incubation with secondary antibodies for 5 minutes at room temperature. Sections were mounted in Mowiol 4–88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich) and visualized on a Zeiss Axiophot fluorescence microscope. Tissue samples from three or more animals were stained for representative data shown (Fig. 1g, h, Fig. 3a, b, and Extended Data Fig. 3a–c, f–h).

Chang A.J., Ortega F.E., Riegler J., Madison D.V, & Krasnow M.A. (2015). Oxygen control of breathing by an olfactory receptor activated by lactate. Nature, 527(7577), 240-244.

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5

Immunofluorescence Staining of Neuronal Markers

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After washing in PBS, sections or fixed cells (with PFA 4%, 10 min at room temperature) were treated with PBS - 0.01% Triton X100 - 10% serum for 30 min. They were then incubated overnight at 4°C with the following primary antibodies diluted in the blocking buffer: rabbit anti-CaMKIIβ (1/300; Abcam, ab34703), mouse anti-CaMKIIδ (1/50, Santa Cruz, sc-100362), mouse anti-CaMKIIγ (1/50, Santa Cruz, sc-517278), rabbit anti-Cux1 (1/50; Santa Cruz, sc-13024), chicken anti-GFP (1/1000; Abcam, ab13970), mouse anti-Ki67 (1/50; BD Pharmingen™, 550609), mouse anti-NeuN (1/500; Millipore, MAB377), rabbit anti-pHH3 (1/200; Upstate, 06-570), rabbit anti-Tbr2 (1/500; Abcam, ab23345), mouse anti-Tuj1 (1/200; Covance, MMS-435P). Sections were then incubated with appropriate fluorescent secondary antibodies. For Cux1 and NeuN immunostainings, a step of antigen retrieval with citrate was performed before the step of blocking (sodium citrate pH=6 for 15 min at 90°C).
F-actin filaments were visualized using rhodamine-labelled phalloidin (Sigma). After fixation, sections or cells were permeabilized 10 min with 0.1% Triton X100 - PBS, incubated with rhodamine-labelled phalloidin diluted in PBS (0.2 μg/ml) for 40 min and washed several times in PBS.

Nicole O., Bell D.M., Leste-Lasserre T., Doat H., Guillemot F, & Pacary E. (2018). A novel role for CAMKIIβ in the regulation of cortical neuron migration: implications for neurodevelopmental disorders. Molecular psychiatry, 23(11), 2209-2226.

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6

Multicolor Immunofluorescence Imaging Protocol

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Tissue was fixed in 4% PFA/PBS at room temperature for 10 minutes and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at −80°C. Sections were cut at 10 µm using a Leica CM3050S cryostat and incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-TH (Abcam, ab112), chicken anti-GFP (Abcam, ab13970), rat anti-CD31 (BD Pharmingen, 553370), chicken anti-β-galactosidase (Abcam, ab9361), and mouse anti-smooth muscle actin (Sigma, A5228) used at 1:500. Mouse anti-smooth muscle actin antibody was directly conjugated to Cy5 NHS ester (GE Healthcare), and unbound dye was removed on a P-30 gel exclusion column (BioRad)³⁵. Incubation with secondary antibodies was 45 minutes at room temperature. Secondary antibodies were conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or DyLight 488 (Jackson ImmunoResearch). Staining with 4’,6-diamidino-2-phenylindole (DAPI; 1 ng/ml, Life Technologies) was performed after incubation with secondary antibodies for 5 minutes at room temperature. Sections were mounted in Mowiol 4–88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich) and visualized on a Zeiss Axiophot fluorescence microscope. Tissue samples from three or more animals were stained for representative data shown (Fig. 1g, h, Fig. 3a, b, and Extended Data Fig. 3a–c, f–h).

Chang A.J., Ortega F.E., Riegler J., Madison D.V, & Krasnow M.A. (2015). Oxygen control of breathing by an olfactory receptor activated by lactate. Nature, 527(7577), 240-244.

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7

Histological and Immunofluorescence Analysis of Mouse Hearts

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The excised
hearts of the sacrificed mice were retrogradely perfused with PBS
to wash the coronary vasculature and LV and fixed with 4% paraformaldehyde
overnight at 4 °C. Each tissue sample was embedded in paraffin.
Sections (2 μm) stained with hematoxylin and eosin and Masson’s
trichrome were used to calculate fibrosis size and wall thickness
using Image Pro version 4.5 (Media Cybernetics, Bethesda, MD). The
sections were subjected to immunofluorescence staining using standard
protocols. Primary antibodies against GFP (abcam ab13970), cTnT, Vimentin
(BD Bioscience BD550513), CD31 (abcam ab28364), collagen type I (abcam
ab21286), and αSA (Sigma A2172) were used to investigate AAV
transduction (GFP), fibroblasts (Vimentin), vascular regions (CD31),
fibrosis (COI), or cardiogenic regions (cTnT or αSA). Primary
antibodies against proliferating cell nuclear antigen (PCNA; Abcam)
and Caspase-3 (Santa Cruz Biotechnology) were used for examining proliferation
and cell death in ischemic tissues. Sections were counterstained with
DAPI (Vector Laboratories, Burlingame, CA) and examined using a FluoView
1000 confocal microscope (Olympus, Tokyo, Japan).

Yoo S.Y., Jeong S.N., Kang J.I, & Lee S.W. (2018). Chimeric Adeno-Associated Virus-Mediated Cardiovascular Reprogramming for Ischemic Heart Disease. ACS Omega, 3(5), 5918-5925.

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8

Immunostaining Analysis of Mammalian Expression Vectors

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Mammalian (pCaggs) expression vectors encoding p27^kip1, p27^kip1(ck-) and RhoA(N19) have been described previously [16 (link)], while an Rp58 expression construct with a pCIG vector which lacks a GFP cassette was used [11 ]. RNAi for Rp58 was achieved using a pool of targeting siRNAs (Dharmacon GE Life Sciences) which was previously verified for specificity of knockdown as well as a pSilCaggs-Rnd2shRNA1 vector to induce Rnd2 RNAi [11 ]. Primary antibodies used for immunostaining analysis include chicken antibody to GFP (Abcam, ab13970, 1:700), mouse anti-p27^kip1 (BD Biosciences, 1:400), rabbit anti-Rp58 (Proteintech Group, 1:250), rabbit anti-Ki67 (NCL-Ki67p, Leica, 1:1000), pHH3(ser10) (06–570, Merck Millipore, 1:1000), mouse anti βIII-tubulin (Covance, MMS-435P, 1:1000), mouse anti-Nestin (Millipore, MAB353, 1:300), rabbit anti-Pax6 (Covance, PRB-2788, 1:500), rabbit anti-Tbr2 antibody (Abcam, ab233345, 1:500), rabbit polyclonal antibody to GFP (Invitrogen, A6455, 1:1000). Alexa fluor secondary antibodies include goat anti- chicken IgG (Invitrogen, A11039, 1:700), goat anti-mouse (Invitrogen, A11031, 1:800), and goat anti-rabbit IgG (Invitrogen, A6455, 1:1000). The nuclei of cells were visualised with DAPI.

Clément O., Hemming I.A., Gladwyn-Ng I.E., Qu Z., Li S.S., Piper M, & Heng J.I. (2017). Rp58 and p27kip1 coordinate cell cycle exit and neuronal migration within the embryonic mouse cerebral cortex. Neural Development, 12, 8.

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Ab13970

Lab products found in correlation

8 protocols using ab13970

Immunostaining of Mouse Embryo Sections

Whole-mount Immunostaining of Telogen Skin

Whole-mount Immunostaining of Telogen Skin

Multicolor Immunofluorescence Imaging Protocol

Immunofluorescence Staining of Neuronal Markers

Multicolor Immunofluorescence Imaging Protocol

Histological and Immunofluorescence Analysis of Mouse Hearts

Immunostaining Analysis of Mammalian Expression Vectors

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