Micropatterned and unpatterned 12 mm TPU coupons (n=3) were pressed to the bottom of a sterile Petri dish around the outer perimeter. Surfaces were immersed in 20 ml of a final concentration of ~106 platelets/μl derived from platelet rich plasma (Bonfils Blood Bank) diluted with PBS supplemented with 10 mM calcium chloride for 2 h 37°C while rotating at 80 rpm. Surfaces were rinsed three times by adding PBS to the dish, rotating (80 rpm) for 30 s on an orbital shaker, and decanting the rinsate. Platelets were fixed on surfaces for 1 h in 1% paraformaldehyde at room temperature. Fixed surfaces were blocked with 10% normal goat serum (Invitrogen) for 1 h before applying a primary αIIbβ3 integrin antibody (ab662, abcam) diluted 1:1000 in 10% normal goat serum (Invitrogen) and incubating overnight at 4°C. Surfaces were exchange rinsed with PBS five times before adding secondary Alexa-Fluor-555 IgG antibody (Invitrogen) diluted 1:100 in 6% normal goat serum for 1 h at room temperature. Images were taken in 10 pre-determined locations per sample by fluorescent microscopy (Zeiss LSM 510 META on Axiovert 200 M), area coverage (μm2) was measured using ImageJ, and each coverage data point was log transformed prior to statistical analysis. Each experiment was replicated four times.
Normal goat serum (ngs)
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Canada, China, Italy
Normal goat serum is a biological material derived from the blood of healthy goats. It is used as a protein supplement and blocking agent in various immunological and cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (96 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (66 mentions)
Bovine serum albumin (bsa), by Merck Group (44 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (34 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (33 mentions)
Hoechst 33342, by Thermo Fisher Scientific (29 mentions)
Paraformaldehyde (pfa), by Merck Group (29 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (21 mentions)
Normal goat serum (ngs), by Merck Group (16 mentions)
Triton x 100, by Thermo Fisher Scientific (15 mentions)
Lsm 710 confocal microscope, by Zeiss (15 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (15 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (13 mentions)
Lsm 710, by Zeiss (12 mentions)
Fluoromount g, by Southern Biotech (11 mentions)
Bx51 microscope, by Olympus (11 mentions)
Tween 20, by Merck Group (11 mentions)
Vectashield, by Vector Laboratories (10 mentions)
Triton x, by Merck Group (10 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (10 mentions)
707 protocols using normal goat serum (ngs)
1
Platelet Adhesion on Micropatterned TPU
2
Immunofluorescence Staining of Organelle Markers
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OSCs in the growth phase were seeded on coverslips for 1 h. After rinsing with PBS, cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature, washed in PBS three times for 5 min, permeabilized with PBTX (PBS with 0.1% Tween-20 and 0.3% Triton X-100) for 20 min, blocked with PBTX containing 3% normal goat serum (Invitrogen) for 1 h at room temperature, incubated with primary antibody in PBTX with 3% normal goat serum for 3 h at room temperature, washed in PBTX three times for 10 min, incubated with secondary antibodies in PBTX with 3% normal goat serum overnight at 4°C and then washed in PBTX three times for 10 min in a dark chamber. Coverslips were mounted with a drop of SlowFade Gold Antifade reagent (Invitrogen) containing DAPI. As primary antibodies we used rabbit polyclonal α-Piwi (1:300; Santa Cruz Biotechnology, sc-98264), mouse monoclonal α-NPC (Mab414, 1:300; ab24609, Abcam), rabbit polyclonal α-Xmas-2 (1:300) (28 (link)), rabbit polyclonal α-Thoc5 (1:300) (29 (link)), rabbit polyclonal α-Elys (1:600; this work), rabbit polyclonal α-Pol II (1:500; ab5408 (4H8), Abcam) and guinea pig polyclonal α-LBR (1:1000) (31 (link)). As a secondary antibodies we used Alexa Fluor 546-conjugated goat α-rabbit IgG (Invitrogen) or Alexa Fluor 488-conjugated goat α-mouse IgG (Invitrogen), or Alexa Fluor 633-conjugated goat α-guinea pig IgG (Invitrogen).
3
Immunofluorescence Analysis of Retinal Cell Structures
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The eyeballs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 12 h. Under an Olympus SZ-STB1 (Olympus) dissection microscope, the anterior segment tissues, vitreous, and retinas were removed to isolate the RCSC. Approximately four to eight relaxing radial incisions were made, and the remaining RCSC were incubated overnight in a blocking solution composed of 5% NGS (Thermo Fisher Scientific) with 0.01% Triton-X (Sigma-Aldrich). The RPE-choroidal-scleral complexes were then incubated with beta-amyloid (36-6900; 1:100; Thermo Fisher Scientific), crystallin alpha B (Ab151722, 1:50; Abcam), Iba1 (019-19741; 1:100; Wako), and Anti-CD4 (MA5-12259, 1:100, Thermo Fisher Scientific). The RPE-choroidal-scleral complexes were then incubated with primary antibody dissolved in 2.5% NGS (Thermo Fisher Scientific) for 24 h before being washed three times for 10 min with PBS-T; then, the samples were incubated for 24 h with Cy5 conjugated secondary antirabbit (ab97077) and antimouse (ab6563) secondary antibody 1:1,000 in 2.5% NGS (Thermo Fisher Scientific) and DAPI 1:5,000 (Sigma-Aldrich). After another PBS-T washing, three times for 10 min, the samples were mounted on slides with ProLong Diamond antifade reagent (Invitrogen).
4
Quantifying Natalizumab Binding Assay
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Wells of a NeutrAvidin-coated 96-well plate or 8-well strips (Thermo Fisher) were coated with 100 µl of biotinylated peptide at 7.5 µg/ml in molecular grade H2O (GE Healthcare Life Sciences, Logan, UT) for 1 h. Wells were washed five times with TBST. Wells were then blocked with 200 µl of 5% normal goat serum diluted from 10% normal goat serum (Thermo Fisher) in TBS for 1 h. Wells were washed five times with TBST. Antibody was spiked into 2.5% BSA in TBST (2.5% BSA/TBST) or into pooled defibrinated human AB serum (serum; Gemini, Woodland CA) diluted in 2.5% BSA/TBST. The final dilution of serum was 0.4% unless otherwise noted. Wells were incubated with 100 µl of antibody-spiked samples for 1 h and then washed five times with TBST. Bound natalizumab was detected with mouse anti-human IgG4 horse radish peroxidase (anti-IgG4-HRP; Abcam, Cambridge, MA) diluted 1:2000-1:5000 in TBST. Wells were incubated with 100 µl of diluted anti-IgG4-HRP for 30 m and then washed nine times with TBST. Turbo TMB substrate (Thermo Fisher) was added to the wells and allowed to develop for 12–15 m shielded from light. The reaction was stopped with 1 M H2SO4 (Thermo Fisher). Optical density (OD) was read at 450 nm on a Multiskan FC plate reader (Thermo Fisher) and data were analyzed with GraphPad Prism v5 or v7 software (Graphpad Software Inc, La Jolla CA).
5
Whole Mount Immunofluorescence Microscopy of PLVs
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PLVs with or without the adjacent SSV were processed for whole mount immunofluorescent microscopy, as previously described [22 ]. Briefly, after dissection the vessels were fixed in 10% normalized buffered formalin (NBF) for 30 min, and then washed in 0.1% Triton X-100 (Millipore Sigma Cat# X100) / 1× TBS (Bio-Rad Cat# 1706435) solution (3×, 10min) rocking at room temperature. The vessels were then permeabilized overnight in 0.3% Triton X-100 / 1× TBS solution rocking at 4 °C. The vessels were blocked in 5% normal goat serum (Thermo Fisher Scientific Cat#50062Z) / 0.3% TritonX-100 / 1× TBS solution rocking at room temperature for 1 h. The vessels were incubated overnight rocking at 4 °C in primary antibody solution diluted in 5% normal goat serum / 0.3% TritonX-100 / 1× TBS; anti-alpha smooth muscle actin (αSMA) Alexa Fluor 488 conjugated antibody (Thermo Fisher Scientific Cat# 53-9760-82) was used at 1:100 dilution. To wash the primary antibody, the vessels were incubated with 0.1% TritonX-100 / 1× TBS (3×, 10 min) then mounted on a microscope slide with 1 drop of ProLong Gold Antifade Mountant (Thermo Fisher Scientific Cat# P36930) and NucBlue Live ReadyProbes (Hoechst 33342 formulation; Thermo Fisher Scientific Cat# R37605). The slides were then imaged by an Olympus VS120 slide scanner or Nikon A1R HD confocal microscope.
6
Immunofluorescence Labeling of pERK1/2 in Mouse Brains
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The whole brains from perfused subject mice were fixed with ice-cold 4% paraformaldehyde (Sigma-Aldrich) in PBS for one day after perfusion. The brain samples were dehydrated in 30% sucrose for three days and embedded in the Frozen Section Compound (Leica, 3801480). The frozen samples were sectioned at 40 µm and blocked with 10% Normal Goat Serum (Thermo Fisher Scientific, PCN5000) in 0.025% Tween 20 solution for 1.5 h. The sections underwent washing twice with 0.025% Tween 20 and once with PBS, followed by labeling with anti-pERK1/2 (1:100, Santa Cruz Biotechnology, SC-7383). For immunofluorescence labeling, the sections were incubated with pERK1/2 in 1/3 of 10% Normal Goat Serum for three days at 4 °C. The sections were then rinsed 3 times with 0.025% Tween 20 and twice with PBS followed by incubation with secondary antibody (Alexa Fluor® 488 Goat Anti-Rabbit IgG, 1:500, Thermo Fisher Scientific, A-11008) in 1/3 10% Normal Goat Serum at 4 °C for 2 h. After rinsing twice with PBS, the nuclei in the sections were labelled with TO-PRO3 (1:1000, Thermo Fisher Scientific, T3605) for 10 min, then washed with PBS. The sections were mounted in the coverslip with ProLong® Gold antifade reagent (Thermo Fisher Scientific, P10144). The images were acquired with a fluorescence microscope (Carl Zeiss, LSM710) and assembled in Adobe Photoshop CS6 (Adobe).
7
Immunostaining of Goblet Cells and Tight Junctions
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To detect goblet cells and tight junctions, we used antibodies targeting Muc5Ac and ZO-1, respectively. The cultures were first fixed with 4% (w/v) paraformaldehyde (Sigma, Saint Louis, United States) for 15 min and then blocked for 1 h in a blocking solution (0.5% Triton X-100, 5% normal goat serum, and 2% bovine serum albumin; all reagents from Thermo Fisher Scientific, Waltham, MA, United States) in 1× Dulbecco’s phosphate-buffered saline (D-PBS; without calcium, magnesium, or Phenol red; STEMCELL Technologies). The cultures were stained with a Muc5AC antibody conjugated to Alexa 550 (1:250, ab218714, Abcam) or a ZO-1 antibody (1:250, 339194, Thermofisher), diluted in D-PBS with 2% normal goat serum (Thermo Fisher Scientific, Waltham, MA, United States) and 1% bovine serum albumin (Thermo Fisher Scientific). Nuclei were counterstained by using ProLong™ Diamond Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific). Images were acquired with the CellInsight™ CX7 HCS platform (Thermo Fisher).
8
Immunostaining of Eye Tissue in Zebrafish
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The procedure of fixation for the eyeballs was the same as described above, but slightly
modified: Whole larvae were fixed at room temperature for 60 min. The eyes of adult
zebrafish were enucleated and fixed in a solution of 4% PFA at 4°C overnight.
Paraffin sections were pretreated with heating at approximately 98°C for 45 min in 10 mM
citrate buffer (pH 6.0). Immunostaining was performed in accordance with the standard
protocols using 5% normal goat serum (Thermo Fisher Scientific, Waltham, MA, U.S.A.) in
0.2% Triton X-100 as a blocking reagent, and primary antibody was diluted with 1% normal
goat serum/0.2% Triton X-100. The secondary antibody was diluted with DAKO Real Antibody
Diluent (Agilent Technologies, Santa Clara, CA, U.S.A.), as follows: The primary antibody
used was anti-PCNA (1:1,000, clone PC10, Sigma-Aldrich), and the secondary antibody used
was Alexa 488-conjugated goat anti-mouse IgG (1:1,000, Thermo Fisher Scientific). Cell
nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Thermo Fisher
Scientific).
modified: Whole larvae were fixed at room temperature for 60 min. The eyes of adult
zebrafish were enucleated and fixed in a solution of 4% PFA at 4°C overnight.
Paraffin sections were pretreated with heating at approximately 98°C for 45 min in 10 mM
citrate buffer (pH 6.0). Immunostaining was performed in accordance with the standard
protocols using 5% normal goat serum (Thermo Fisher Scientific, Waltham, MA, U.S.A.) in
0.2% Triton X-100 as a blocking reagent, and primary antibody was diluted with 1% normal
goat serum/0.2% Triton X-100. The secondary antibody was diluted with DAKO Real Antibody
Diluent (Agilent Technologies, Santa Clara, CA, U.S.A.), as follows: The primary antibody
used was anti-PCNA (1:1,000, clone PC10, Sigma-Aldrich), and the secondary antibody used
was Alexa 488-conjugated goat anti-mouse IgG (1:1,000, Thermo Fisher Scientific). Cell
nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Thermo Fisher
Scientific).
9
Immunofluorescence Staining of Adherent Cells
10
Immunofluorescence Analysis of YAP Localization
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