For assessment of fluorescence, live parasite samples were assessed, and images captured, on a Zeiss LSM510 confocal microscope. ER-ID Red (Enzo Life Sciences, United Kingdom) was used to stain the ER according to the manufacturer’s instructions. Parasites were prepared for electron microscopy by overnight fixation in 2.5% glutaraldehyde/2.5% paraformaldehyde/0.1 M Na cacodylate buffer at 4 °C. Samples were post-fixed with 1% osmium tetroxide/0.1 M Na cacodylate buffer, washed with buffer followed by MilliQ water, en bloc stained with 3% aqueous uranyl acetate, dehydrated in ascending ethanol concentrations, rinsed briefly in propylene oxide, then embedded and polymerised in Taab epoxy resin. Ultrathin sections were cut and mounted on Pioloform-coated copper grids and stained with lead citrate. Immunogold labelling was carried out as previously described (McDonald et al., 1995 (link)) using rabbit polyclonal antibody to GFP (ab6556; Abcam) diluted 1:500 and goat-anti-rabbit IgG 10 nm gold-conjugated (BB International, United Kingdom) diluted 1:400. Samples were examined on a Jeol 1200EX Mark II transmission electron microscope and digital images recorded with a 1 K 1.3 M pixel High Sensitivity AMT Advantage ER-150 CCD camera system.
Er id red
Manufactured by Enzo Life Sciences
Sourced in United Kingdom, United States
ER-ID Red is a fluorescent probe designed for the detection and quantification of endoplasmic reticulum (ER) in live cells. It selectively stains the ER and can be used in various cell-based assays and imaging applications.
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Lab products found in correlation
Golgi id green, by Enzo Life Sciences (2 mentions)
Mito id red, by Enzo Life Sciences (2 mentions)
Ab6556, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lyso id red, by Enzo Life Sciences (1 mentions)
Fixation medium a, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
4 protocols using er id red
1
Fluorescence and Ultrastructural Analysis of Parasites
2
ER Expansion Measurement by Flow Cytometry
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ER expansion was assessed using ER-ID Red (Enzo Life Sciences Farmingdale, NY, USA) staining. Briefly, 4 × 105 cells/well were seeded into 6-well plates and treated with various concentrations of PT for 24 h. Subsequently, the treated cells were harvested and stained with ER-ID Red reagent for 30 min at room temperature. A FACSCalibur flow cytometer (Becton, Dickinson, and Company, San Jose, CA, USA) and Cell Quest Pro software were used to analyse the stained cells.
3
Antibody Panel for Vesicular Trafficking
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Antibodies to caveolin-1 (D46G3), clathrin HC (D3C6), Rab5 (C8B1), APPLI (D83H4), EEA1 (C45B10), Rab9A (D52G8), Rab11 (D4F5) and syntaxin-6 (C34B2) rabbit antibodies were from Cell Signaling Technology. MITO-ID Red, Golgi ID Green, ER ID Red and Lyso ID Red were purchased from Enzo Life Sciences. Anti-α3 integrin (CD49c) and APC-anti-α3 integrin (CD49c) antibody (clone ASC-1), anti-CD9 antibody and FITC anti-CD9 antibody (clone H19a), PE anti-human IGF-1R (clone 1H7/CD221), PE anti-human CD115 (clone 9-4D2-1E4), PE anti-human EGFR (AY13), anti-CD59 antibody and PE anti-CD59 antibody (p282), anti-CD276 antibody and APC anti-CD276 antibody (MIH42), anti-CCR2 antibody (K036C2) and anti-CXCR4 antibody (12G5) were from Biolegend. Anti-CXCR2 antibody and APC anti-CXCR2 (Clone 6C6) were from BD Biosciences. Anti-TIMP3 antibody was from Biorbyt Ltd. (Cambridge, United Kingdom). Alexa 555 goat anti-IgG H+L antibodies were from Life Technologies. Alexa 647 anti-P2Y11 (505214) and Alexa 647 anti-MRGX2 (477533) were from R&D Systems. Anti-human LL-37 antibody (1C12) was from Hycult Biotch. Goat anti-mouse IgG (H&L):15 nm Gold was from BBI Solutions. LL-37 was synthesized by the standard FMOC chemistry, purified by reversed phase HPLC, and the mass verified by nanospray mass spectrometry. The concentration was determined by amino acid analysis (Zhang et al., 2008 (link)).
4
Multiparametric Analysis of Protein and Organelle Dynamics
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For HC and LC protein, cells (1 x 10 6 ) were fixed using a 1:1 solution of Fixation Medium A (Invitrogen) and flow cytometry buffer (PBS with 5% BSA) for 15 min at RT and then incubated with Permeabilisation Medium B (Invitrogen) containing 1:20 goat f(ab')2 antihuman IgG Alexa Fluor conjugated to 488 (Invitrogen, excitation/emission: ~500/520 nm) and goat f(ab')2 anti-human kappa conjugated to APC (Biolegend, excitation/emission: ~650/675 nm) for 15 min, before being washed and analysed. HC and LC mRNA were investigated using a PrimeFlow RNA kit with custom probes (Affymetrix, Merck) following the manufacturer protocol. Quantitation of organelles was performed by staining with Golgi-ID Green (excitation/emission: ~450/530 nm), ER-ID red (excitation/emission: ~580/660 nm), Mito-ID red (excitation/emission: ~558/690 nm; all from Enzo Life Sciences) and MitoTracker Deep Red (Invitrogen, excitation/emisssion: ~644/665 nm) using the manufacturers' protocols. DAPI (excitation/emisssion: ~340/450 nm) or Sytox nuclear (excitation/emission: ~444/480) counter-stain were used for nuclear localization.
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