100,000 cells were seeded on glass slides (Falcon) and incubated for 2 days. Cells were washed with PBS, fixed with ice-cold acetone, and washed with TBST. Endogenous peroxidase was blocked (1% H2O2, 10 minutes), cells were washed twice with TBST, and incubated in 5% normal goat serum for 1h. Antibodies were diluted in Antibody Diluent (Cell Signaling Technologies) at a concentration of 1:800 and incubated at 4 °C overnight. Cells were washed twice with TBST and Signal Stain Boost IHC Detection Reagent was added (Cell Signaling Technologies). After washing with TBST, Signal Stain DAB substrate was added for 45 s. Thereafter, cells were washed with TBST. Counterstaining was performed with Haematoxylin (Sigma Aldrich).
Glass slide
Manufactured by BD
Sourced in Italy
A glass slide is a thin, flat piece of glass used in various laboratory applications. Its primary function is to provide a transparent surface for mounting and viewing samples, such as cells, tissues, or other microscopic specimens, under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
E ab 17922, by Elabscience (2 mentions)
E ab 1055, by Elabscience (2 mentions)
E ab 18031, by Elabscience (2 mentions)
Antibody diluent, by Cell Signaling Technology (1 mentions)
Signalstain boost ihc detection reagent, by Cell Signaling Technology (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fibronectin coated, by BD (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Echo revolve microscope, by Echo Medical Systems (1 mentions)
E ab 1015, by Elabscience (1 mentions)
7 protocols using glass slide
1
Immunocytochemistry Protocol for Cultured Cells
2
Immunostaining of OATP2A1 in HUVECs
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OATP2A1 was immunostained as described previously48 . HUVECs were plated on glass slides (BD Falcon, Franklin Lakes, NJ) at a density of 5 × 104 cells/0.7 cm2. The cells were fixed in 4% paraformaldehyde, and permeabilized with 0.01% (w/v) Triton X100 in PBS. Immunoreaction was performed by incubating the cells with a 1:200 dilution of anti-human OATP2A1 rabbit polyclonal antibody for 3 hrs at rt, followed by staining with a 1:400 dilution of AlexaFluor® 594-conjugated anti-rabbit IgG (Life Technologies) for 1 hr at rt. The cells were counterstained with Hoechst 33342 (2 μg/mL) for nucleus (blue), and then mounted with Vectashield® (Vector Laboratories, Peterborough, UK). Fluorescence was examined by the use of a confocal laser microscope (LSM710, Carl Zeiss, Göttingen, Germany).
3
Immunofluorescence Microscopy of Cells
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Cells on fibronectin-coated (10 µg/ml) glass slides (BD Biosciences) were fixed in 4% paraformaldehyde (PFA), permeabilised with 0.2% Triton X-100 and blocked (1 h, room temperature) in phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA), prior to incubation (30 min, 37°C) with appropriate antibodies in PBS/0.2% BSA, and Alexa-conjugated secondary antibodies (1 h, room temperature). In some cases cells were stained with fluorescently conjugated antibodies in culture media prior to fixation. Alternatively, actin was stained in permeabilised cells with Alexa488-phalloidin (1 µg/ml; Molecular Probes). For cell rounding assays, IIIA4 mAb (1.5 or 3.0 µg/ml), cross-linked with anti-mouse IgG (Jackson ImmunoResearchs), was added to cells 10 min prior to analysis. Coverslips were mounted onto microscope slides with Fluoromount (Sigma). Fluorescence images were taken on a Leica SP5 microscope and analysed using AnalySIS software (Soft Imaging System, Germany).
4
Corneoscleral Tissue Sectioning and Staining
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OCT-embedded corneoscleral specimens were cut in 5-µm serial sections (CM3050 cryostat; Leica Microsystems, Rijswijk, Netherlands), placed onto glass slides (BDH, Milan, Italy), quickly air-dried, and stored until specific staining. Sections were postfixed in cold 0.05% buffered formaldehyde and used as reported below. Sections from paraffin-embedded specimens were used for basal histology after dewaxing and rehydrating steps (downscaling Et-OH steps until water and buffered saline).
5
Immunofluorescent Staining of HLA-C
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3D Sw71 models or 2D Sw71 monolayers were cultured for 24–48 h in sterile culture chambers on a glass slide (Falcon). Trophoblast derived explants were cultured in the same type of chambers for expansion of EVTs, and after that the explants were removed carefully. The cells were fixed in 2% paraformaldehyde (PFA) in PBS, overnight (ON) at RT and stained for HLA-C using indirect immunofluorescent method. After washing with PBS, the cells were incubated with 10% goat serum (to block the non-specific binding) and then subsequently with purified polyclonal rabbit anti-human HLA-C antibody (E-AB-17922, Elabscience) and AF488-conjugated goat anti-rabbit antibody (E-AB-1055, Elabscience). Negative controls were prepared by omitting primary antibody and/or secondary antibody. Control staining was done with mouse polyclonal anti-human HLA-G antibody (E-AB-18031, Elabscience) and goat anti-mouse IgG (E-AB-1015, Elabscience). The slides were imaged with ECHO Revolve microscope (RVL-100-M, Echo, San Diego, CA, USA).
6
Reticulocyte Age Determination via Dual Staining
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To evaluate the age of the reticulocytes that contained parasites, we used a double staining method, which simultaneously stains the reticulocyte and the parasite. 5 μL of packed infected cells were washed with PBS, mixed with an equal volume of new methylene blue (NMB) (Sigma) and allowed to stain for 15 minutes. Following the staining, the cells were smeared on a glass slide (Falcon) and air-dried. Slides were then methanol fixed and stained with Giemsa. In the resulting slides both the reticulum of the reticulocytes and the nuclear and cytoplasmic material of the parasites were stained (see S1 Fig , Fig 2 ).
7
Immunofluorescence Analysis of HLA-G and HLA-C in 2D and 3D Sw71 Cell Models
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Sw71 and Sw71-Tw-KO cells as monolayer and spheroids were used in this study. The characterization of these cells has been reported in numerous publications (49) (50) (51) . 3D Sw71 models or 2D Sw71 monolayers were cultured for 24-48 hrs in sterile culture chambers on a glass slide (Falcon). The cells were fixed in 2% paraformaldehyde (PFA) in PBS, overnight (ON) at RT and stained for HLA-G and HLA-C using indirect immunofluorescent method. After washing with PBS, the cells were incubated with Super Block (SkyTec Laboratories, Logan, UT, USA) to suppress the non-specific binding. As primary antibodies we used anti-human rabbit polyclonal antibodies against HLA-G (E-AB-18031, Elabscience) and HLA-C (E-AB-17922, Elabscience) in appropriate dilutions for overnight incubation at 4°C. As a secondary antibody goat anti-rabbit IgG conjugated with AF488 (E-AB-1055, Elabscience) was applied. Negative controls were prepared by omitting primary antibody and/or secondary antibody.
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