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).
E ab 1055
Manufactured by Elabscience
The E-AB-1055 is a laboratory instrument designed for the quantitative detection and analysis of target analytes. It utilizes electrochemical techniques to measure and quantify the presence and concentration of specific molecules or compounds in a sample. The core function of the E-AB-1055 is to provide accurate and reliable data for various analytical applications.
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Lab products found in correlation
Glass slide, by BD (2 mentions)
E ab 17922, by Elabscience (2 mentions)
E ab 18031, by Elabscience (2 mentions)
Echo revolve microscope, by Echo Medical Systems (1 mentions)
E ab 1015, by Elabscience (1 mentions)
Fv3000, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (1 mentions)
Cm1860, by Leica (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Mtor 7c10, by Cell Signaling Technology (1 mentions)
5 protocols using e ab 1055
1
Immunofluorescent Staining of HLA-C
2
Immunofluorescent Adipocyte Staining Protocol
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The adipocytes were fixed in 4% paraformaldehyde for 15 min. Then, 0.5% Triton X-100 was used to permeabilize the cells for 10 min, and 5% bovine serum albumin was used to block the cells for 30 min. The samples were incubated with the specific antibody overnight, and then with the corresponding species of fluorescent-conjugated secondary antibody (#E-AB-1055, Elabscience) for 30 min using DAPI (#AR1176, BOSTER). The staining was detected under a laser scanning confocal microscope (FV3000, Olympus).
3
Examining Brain Pathology in CLN7-KO Mice
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Nine-month old CLN7-KO and WT mice were deeply anesthetized with isoflurane and then perfused with chilled PBS, followed by 4% paraformaldehyde in PBS. Then, the brains were removed and transferred into a series of sucrose solutions (10, 20, and 30% sucrose) until they sank at 4°C. Subsequently, 40-μm-thick coronal brain sections were obtained using a freezing cryostat (Leica, CM1860). The brain slices for autofluorescence detection (fig. S16, B and C) were mounted on microscope slides with fluorescent mounting medium for subsequent confocal imaging. The hippocampal slices for CLN7 immunostaining (fig. S16A) were washed in PBS and blocked overnight at 4°C in PBS containing 6% goat serum (v/v), 1% bovine serum albumin (BSA) (w/v), and 0.2% Triton X-100. Then, the slices were incubated with anti-MFSD8 antibodies (1:100: Atlas Antibodies, HPA044802) at 4°C for 3 days in PBS containing 3% goat serum (v/v), 0.2% Triton X-100, and 0.5% BSA (w/v). After washing three times with PBS (10 min each time), brain sections were incubated with the secondary antibody, fluorescein isothiocyanate–conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) (1:200; Elabscience, E-AB-1055), for fluorescent detection.
4
Immunofluorescence Analysis of mTOR and LAMP2 Colocalization
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Immunofluorescence and co-localization analysis were used in Fig 5j and 5k . Cells were plated onto poly-L-lysine-coated confocal dishes. After treatments, cells were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) at room temperature for 10 min and washed/permeabilized with 1× PBST solution (1× PBS and 0.1% Triton X-100) for another 15 min. Then, the cells were blocked with blocking solution (1× PBS with 1% BSA) at room temperature for 30 min. Staining was performed with the indicated primary antibodies in blocking solution (1:200 dilution) at 4°C overnight. The cells were stained with cross-adsorbed secondary fluorescent antibodies (1:100 in blocking solution). For the merged images, Elab Fluor 594 (E-AB-1059, Elabscience, for anti-LAMP2 IFs) and Elab Fluor 488 (E-AB-1055, Elabscience, for anti-mTOR IFs) are shown in magenta and cyan, respectively. Imaging was performed with a 100× oil immersion objective on Olympus confocal microscope. The antibodies used included LAMP2 (66301-1-Ig, Proteintech), mTOR (7C10) (2983, Cell Signaling).
To quantify the co-localization of mTOR with the lysosomal marker LAMP2, the Fiji software was used. Forty individual cells from independent fields were selected for the analysis. The Coloc2 plugin was used to calculate the Pearson’s correlation coefficient.
To quantify the co-localization of mTOR with the lysosomal marker LAMP2, the Fiji software was used. Forty individual cells from independent fields were selected for the analysis. The Coloc2 plugin was used to calculate the Pearson’s correlation coefficient.
5
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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