The placenta tissues were embedded in paraffin and sectioned into 4‐μm slides. Then, slides were deparaffinized and hydrated. After inactivating endogenous peroxidase in 3% hydrogen peroxide, slides were retrieved in citric acid buffer (pH 6.0) by microwaving for 15 minutes. Slides were blocked in normal goat serum (Invitrogen) for 30 minutes at room temperature and incubated with an anti‐Ki67 antibody (Abcam) overnight at 4°C. Samples were then washed with TBST (Invitrogen) and incubated with the appropriate secondary antibody (Abcam) for 2 hours at 37°C. Sections were then washed with TBST and stained using a DAB Detection Kit (Solarbio, Beijing, China). Finally, sections were counterstained with hematoxylin. Section images were captured and analysed using Motic Images Advanced 3.2 software (Motic, XiaMen, China).
Tbs t
Manufactured by Thermo Fisher Scientific
Sourced in United States
TBS-T is a buffered saline solution commonly used in various laboratory techniques, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs). Its core function is to provide a stable and consistent buffer environment for the handling and processing of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Tbs t, by Merck Group (3 mentions)
Tween 20, by Carl Roth (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
Antigen retrieval buffer ph 6, by Abcam (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Cell Signaling Technology (2 mentions)
Immobilin p pvdf membrane, by Merck Group (2 mentions)
Nupage, by Thermo Fisher Scientific (2 mentions)
Tris buffered saline (tbs), by Thermo Fisher Scientific (2 mentions)
Appropriate secondary antibody, by Abcam (1 mentions)
Dab detection kit, by Solarbio (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Images advanced 3, by Motic (1 mentions)
Chemiluminescent reagent, by Cytiva (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
M iggκ bp hrp sc 516102, by Santa Cruz Biotechnology (1 mentions)
Bradford method, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
14 protocols using tbs t
1
Immunohistochemical Analysis of Placental Ki67
2
Western Blot Analysis of LGR5 Protein
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RIPA (Radio-Immunoprecipitation Assay) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract proteins from cells, and protein concentration was established by Bradford method (Sigma–Aldrich, Madrid, Spain). Separation of proteins was carried out in 8% SDS-PAGE gels in which 40 µg of proteins of each sample were loaded previously heated at 65 °C for 20 minutes. Then, proteins were electrotransferred to a nitrocellulose membrane (45 µm pore size) (Millipore, Burlington, MA, USA) and blocked for 1 hour in 5% milk in TBS with 0.1% Tween-20 (TBS-T) (Bio-Rad, Hercules, CA, USA). After several washes in TBS-T, the incubation of the anti-LGR5 primary antibody (1:750) (Anti-LGR5 (OTI2A2): MA5-25,644; Invitrogen, Waltham, Massachusetts, USA) was performed overnight at 4 °C. Then, a secondary antibody (m-IgGκBP-HRP: sc-516,102; Santa Cruz Biotechnology, Dallas, Texas, EEUU) was added (1:2000) for 1 hour at room temperature. Chemiluminescent reagents (Amersham Biosciences, Saint Louis, MO, USA) were used for the detection of membrane-bound antibodies. Besides, anti-β-actin IgG (A3854, Sigma Aldrich, Madrid, Spain) (1:25,000 dilution) was used as an endogenous control. The open-source Fiji image analysis software was used to quantify the bands obtained to calculate relative protein expression.28 (link)
3
Dot Blot Assay for α-Synuclein Oligomers
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Dot Blot assay with recombinant α-syn oligomers was performed as previously described [25 (link)]. Briefly, 0.2 – 2 μg α-syn oligomers were applied to a nitrocellulose membrane (pore size 0.22 μm, Whatman Protran, Sanford, ME, USA), blocked with 10 (w/v)% non-fat milkin Tris-buffered saline (TBS) containing 0.01 (v/v) % Tween 20 (TBS-T). The membrane was blocked with 10% (w/v) non-fat dried milk in TBS-T (Sigma-Aldrich, St. Louis, MO, USA) at RT for 1 h. After three washes with TBS-T, the membrane was incubated with anti-oligomer antibody A11 (1:100; Invitrogen, Carlsbad, CA, USA) or Syn-1 antibody (1:1000; BD transduction, Franklin Lakes, NJ, USA) overnight at 4 C with gentle agitation. The membranes were then washed three times for 5 min with TBS-T, incubated with horseradish peroxidase conjugated anti-rabbit IgG or anti mouse IgG (1:2000; Jackson Immuno Research Laboratories, Baltimore, PA, USA) in 5 (w/v) % non-fat dried milk in TBS-T and incubated for 1 h at RT. The blots were washed three times with TBS-T and developed with Pierce ECL chemiluminescence kit from Thermo Scientific (Rockford, IL, USA).
4
Immunohistochemical Staining of Myocardial Tissue
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Sections of 4 µm myocardial tissue were created from paraffin-embedded formalin-fixated samples. Sections were deparaffinized and rehydrated, followed by antigen retrieval by incubation at 95 °C in Antigen Retrieval Buffer pH 6.0 (Abcam, Cambridge, UK). Samples were then blocked in TRIS-buffered saline with 0,1 % (v/v) Tween-20 (NaCl, TRIS, and Tween-20 (Roth, Karlsruhe, Germany) with 5 % (v/v) normal goat serum (Invitrogen, Carlsbad, CA, USA) for 1 h. Slices were incubated in blocking buffer with the primary antibodies at 4 °C overnight (CD47 (ab218810), rabbit, 1:2000, Abcam, Cambridge, UK; CD31 (DIA-310), rat, 1:20, Dianova, Hamburg, Germany) followed by staining with secondary antibodies at room temperature for 1 h (anti-rabbit AlexaFluor680, 1:200; anti-rat Cy3, 1:200, both Invitrogen, Carlsbad, CA, USA). Nuclei were stained with DAPI 1:5000 (1:5000 in TBS-T, Invitrogen, Carlsbad, CA, USA) followed by preservation in Prolong gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Images were acquired using an Olympus BX51 microscope (Olympus, Shinjuku, Japan) and processed using ImageJ software.
5
Whole Fish Embryo Immunostaining
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Whole fish embryos were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and stored in 100% methanol at −20 °C. After rehydration with 50% methanol and blocking with 1% bovine serum albumin (FUJIFILM Wako Pure Chemical Co. Osaka, Japan) in TBS containing 0.05% Tween 20 (TBS-T) (Sigma-Aldrich Co. St. Louis, MI, USA) to reduce non-specific binding, embryos were incubated separately with anti-beta dystroglycan (1:100, Novocastra. Wetzlar, Germany), anti-laminin (1:50, Sigma-Aldrich Co. St. Louis, USA), and anti-myosin heavy chain antibodies (F59, 1:25, Santa Cruz Biotechnology. Dallas, TX, USA) at 4 °C overnight. After several washes with TBS-T, the samples were incubated with secondary antibodies (1:500, anti-mouse Alexa Fluor 488, 1:500, anti-rabbit Alexa Fluor 568, Invitrogen Co. Carlsbad, CA, USA) and DAPI (DAKO, Carpinteria, CA, USA) for 30 min. The stained embryos were observed under a confocal microscope (ZEISS. Oberkochen, Germany).
6
Immunofluorescence Staining of MSCs
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MSC-Sal were washed several times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature (RT). Another 5 min of fixation with acetone at RT was performed. Subsequently, cells were incubated with 10% bovine serum albumin (BSA) (Carl Roth GmbH, Karlsruhe, Germany) in Tris-buffered saline 200 mM Tris-base (pH 8), 8% NaCl and 1% Tween-20 (TBS-T) (all from Sigma-Aldrich). Incubation of MSC in TBS-T containing 1% BSA plus a mouse monoclonal antibody against α-tubulin (1:500; Sigma-Aldrich) was followed for 10 h. After three washing steps with TBS-T (each step 5 min), MSC were incubated for 1 h in 1% BSA at RT with Alexa 488 goat anti-mouse secondary antibody 1:500 (Gibco Invitrogen) and 5 mg/ml DAPI (Sigma-Aldrich). A fluorescence microscope (Leica DMI 4000B Inverted Microscope) was used for cell examination.
7
GeoMx DSP Tissue Immunostaining Protocol
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FFPE tissue sections (5 µm thick) were processed according to the GeoMx® Digital Spatial Profiler (DSP) protocol (NanoString Technologies, Inc.). Briefly, tissue was deparaffinized and rehydrated with CitriSolv (Decon Labs), ethanol and ddH2O washes at room temperature. Antigen retrieval was performed in citrate buffer pH 6.0 (Millipore) at ~ 115 °C under high pressure and washed in tris-buffered saline with Tween 20 (TBS-T) (Cell signaling Technology). Tissue was blocked with Buffer W (Nanostring) for 1 h at room temperature. Slides were incubated at 4 °C overnight with antibody mixture including fluorescently tagged antibodies for IBA1-AF532 (Nanostring Technologies, Inc. #121,300,306) and CD45-AF594 (NanoString Technologies, Inc. #121,300,301) and the following oligo-tagged NanoString GeoMx human detection antibody mixtures: Immune Core Profiling, Immune Cell Typing, Immune Activation, and Cell Death (see Supplemental Material for list of detection targets). Slides were washed with TBS-T, fixed with 4% formaldehyde (Invitrogen) for 1 h at room temperature, then re-washed with TBS-T. Nuclei were stained with SYTO 13 (NanoString).
8
Immunofluorescent Staining of Myocardial Tissue
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Sections of 4 µm myocardial tissue were created from paraffin-embedded formalin-fixated samples. Sections were deparaffinized and rehydrated, followed by antigen retrieval by incubation at 95 °C in Antigen Retrieval Buffer pH 6.0 (Abcam, Cambridge, UK). Samples were then incubated in 3% H2O2 for 10 min, followed by blocking in TRIS-buffered saline with 0,1% (v/v) Tween-20 (NaCl, TRIS, and Tween-20 (Roth, Karlsruhe, Germany) with 5% (v/v) normal goat serum (Invitrogen, Carlsbad, CA, USA) for 1 h. Slices were incubated in blocking buffer with the primary antibodies at 4 °C overnight (PDL1 (ab213480), rabbit, 1:50, Abcam, Cambridge, UK; CD31 (DIA-310), rat, 1:20, Dianova, Hamburg, Germany; or cardiac troponin T (MA5-12960), mouse, 1:50, Invitrogen, Carlsbad, CA, USA) followed by staining with secondary antibodies at room temperature for 1 h (anti-rabbit AlexaFluor680, 1:200; anti-rat Cy3, 1:200; or anti-mouse AlexaFluor594, all Invitrogen, Carlsbad, CA, USA). Nuclei were stained with DAPI 1:5000 (1:5000 in TBS-T, Invitrogen, Carlsbad, CA, USA) followed by preservation in Prolong gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Conventional hematoxylin and eosin stainings were created from slices from the same specimens. Images were acquired using an Olympus BX51 microscope (Olympus, Shinjuku, Japan) and processed using ImageJ software.
9
Protein Expression Analysis of VSMCs
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After stimulation for 4 days, total proteins were extracted from the VSMCs, and the concentrations were measured with the BCA protein assay kit. The samples were mixed with loading buffer and boiled for 5 min. The protein samples were resolved on 10% SDS‐PAGE gel (70 V, 120 min) and electro‐transferred to a PVDF membrane (15 V, 30 min) and blocked with 5% non‐fat dry milk in TBS‐T [20 mmol/L Tris‐HCl (pH 7.6), 150 mmol/L NaCl, and 0.02% Tween 20] (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature with agitation. Subsequently, the primary antibodies of Kca3.1 (1:300), Runx2(1:200), and GAPDH (1:500) were added to the samples, respectively, and incubated at 4°C overnight. After 1 h incubation with the secondary antibody (1:2000, Jackson ImmunoResearch), the samples were visualized using ECL reagent (Pierce) and imaged. The images were analyzed by using Chemiluminescence detection System. GAPDH was used as an endogenous control. The experiments were replicated three times.
10
Dose-Dependent Histone H2A.X Phosphorylation
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