Following the dewaxing, hydration and antigen retrieval, the slides were incubated with 3% H2O2 for 20 min to quench endogenous peroxidase enzymes and 1% bovine serum albumin (BSA, A3608, Sigma) blocking buffer for 1 h to reduce nonspecific binding. Primary antibodies against CD68 (ab125212, Abcam), iNOS (PA1-036, Thermo Fisher Scientific), CD206 (ab64693, Abcam), Podoplanin (E11) (ab10288, Abcam), DMP1 (ab103203, Abcam), HES1 (ab108937, Abcam) were applied at 1:100 dilutions and incubated at 37 °C for 2 h, and rabbit immunoglobulin G (IgG, ab171870, Abcam) was regarded as primary antibody for isotype control. This process was followed by incubation with biotinylated swine-anti-mouse, rabbit, goat secondary antibody (DAKO) for 45 min, and diaminobenzidine (DAB) solution (DAKO) was then applied to visualize the protein-antibody complex. The slides counterstained with Mayer's hematoxylin. Images of all slides were captured using Axion software under the light microscope (Carl Zeiss Microimaging). The immunohistochemistry (IHC) images were quantified using ImageJ software by calculating the percentage of positive cells per field of view (FOV), and 5 FOVs of each sample were calculated.
A3608
Manufactured by Merck Group
Sourced in United States
A3608 is a laboratory centrifuge designed for general-purpose applications. It features a digital display, adjustable speed and time controls, and a rotor capable of accommodating various sample tubes. The core function of this product is to separate components of a liquid mixture based on their density differences through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Ab171870, by Abcam (1 mentions)
Ab10288, by Abcam (1 mentions)
Ab108937, by Abcam (1 mentions)
Ab103203, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Diaminobenzidine dab solution, by Agilent Technologies (1 mentions)
Pa1 036, by Thermo Fisher Scientific (1 mentions)
Spark 20m, by Tecan (1 mentions)
L1395, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Gated stimulated emission depletion microscopy gsted, by Leica (1 mentions)
P9416, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using a3608
1
Immunohistochemical Evaluation of Cellular Markers
2
Quantifying Protein in Small Extracellular Vesicles
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Coomassie assay was used for detection of protein amounts in small EVs. As standard, a serial dilution of 25 μg/mL to 2.5 μg/mL bovine serum albumin (A3608, Sigma-Aldrich, St. Luis, MO, USA) in PBS–/– was used. Each 30 μL of sample/standard and Coomassie assay reagent (23200, Thermo Fisher Scientific, Waltham, MA, USA) were added to wells in a 384 well plate (781061, Greiner Bio-One, Kremsmuenster, AUT) and gently mixed for 30 s at a plate shaker. After an incubation period of 10 min at room temperature in dark conditions, absorbance was measured at 595 nm using a TECAN SPARK 20M (TECAN, Morrisville, NC, USA). AutoOptical density values of samples were compared to the standard curve generated with known concentrations of protein.
3
ECM Adherence Assay for Cell Spreading
4
ECM Adherence Assay for Cell Spreading
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ECM adherence assays were performed as previously described.37 Dishes were incubated with 1 mg/mL collagen 1, 1 mg/mL laminin, or PBS overnight at 4°C, followed by incubation with heat denatured BSA solution (10 mg/mL, Sigma-Aldrich, A3608) for 30 minutes, before rinsing in PBS and plating cells. Plates were fixed with 5% glutaraldehyde for 30 minutes and cells were counterstained with 1% crystal violet solution in distilled water and imaged. The surface area of cells was determined in ImageJ by automatic cell detection after thresholding. For integrin β1–blocking antibody experiments, cells were incubated in media containing 10 μg/mL blocking antibody or equivalent concentration non-targeting isotype control, as above.
5
Yeast Cell Attachment Preparation
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For the first batch of experiments (ncells = 11), a Petri dish (Ibidi) was gently scratched in different directions with sharp tweezers as described (Supplementary Fig. 12a ). After that step, the dish was washed with dish soap, rinsed with ultrapure water and dried with nitrogen. In order to prevent the yeast from attaching to the bottom of the dish, the dish was filled with a solution of 2% bovine serum albumin (BSA, A3608 Sigma-Aldrich) in PBS for 30 min at 37 °C, to adsorb BSA on the bottom of the dish. For most experiments (ncells = 27), the above described scratching has been skipped since with gaining experience the direct pickup of yeast cells from the untreated Petri dish became possible. To prepare the yeast cells for their attachment to the cantilever of the picobalance, the Petri dish was rinsed with PBS and filled with 1.5 ml of pre-conditioned medium at 30 °C. Finally, 20 µl of the yeast culture prepared as described above was diluted into the Petri dish. The Petri dish was then mounted in the controlled environmental chamber of the picobalance, where culture conditions were maintained at 30 °C.
6
Lectin-Mediated Cell Attachment Assay
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Channel slides (µ-Slide VI 0.4, ibidi) were treated with lectin (Sigma-Aldrich, L1395) (0.1 mg/mL in water) for ~5 min, washed with YE5S, and cells were added and incubated for ~5 min to allow for attachment. Unattached cells were removed by washing with YE5S. Solutions of BSA (Sigma-Aldrich, A3608) in YE5S were made fresh. Attached cells in the chamber were first imaged in YE5S medium, then shifted transiently to YE5S containing different concentrations of BSA.
7
MCT4 Protein Localization in Fibroblasts
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1.5 × 105 hTERT-BJ1 fibroblasts per well were seeded in coverslips in 12 well plates. When cells were attached, drug treatments were added for 72 h in triplicate. Cells were fixed with 2% paraformaldehyde (28908, Thermo Scientific) in PBS for 20 minutes at RT, permeabilised in −20°C-cold methanol for 5 minutes at 4°C, quenched with 50 mM NH4Cl in PBS for 10 minutes, rinsed and blocked with IF buffer consisting of 1% BSA (A3608, Sigma) plus 0.1% Tween 20 (P9416, Sigma) in PBS for 1h. Cells were then incubated for 1h with the MCT4 primary antibody (sc-50329, Santa Cruz). The fluorescent secondary antibody was added for 30 minutes. Cells were counterstained with DAPI and samples were mounted using Prolong Gold anti-fade reagent (P36934, Invitrogen). Immunofluorescence pictures were taken in a Leica gated Stimulated Emission Depletion Microscopy (gSTED) with additional confocal and multi-photon illumination (room rg106).
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