Adherent HUVEC cells in culture flasks were washed three times with RPMI. Cells were fixed in culture flasks with 2 percent formaldehyde in RPMI for 2 hours at room temperature, then in 2 percent formaldehyde, 0.0075 percent glutaraldehyde in 1x PBS overnight at 4°C. Fixed cells were washed three times with PBS and blocked with 1X PBS, 3 percent BSA, 0.3 percent Triton X-100 for 1 hour at room temperature. Parasite-derived proteins were detected with either P. falciparum-specific anti-MSP1 (ATCC/MR4) or anti-aldolase (Abcam, Cambridge, UK) primary antibody. Cells were incubated with primary antibody diluted in dilution buffer (1X PBS, 1 percent BSA, 0.3 percent Triton X-100, 0.01 percent sodium azide) for 1 hour at room temperature, and then washed three times with PBS. Alexa Fluor 546 anti-rabbit IgG (Invitrogen, Grand Island, NY) secondary antibody was diluted 1:1000 in dilution buffer, added to cells, followed by incubation in the dark for 1 hour at room temperature. Cells were washed three times with PBS. Prolong Gold Antifade Reagent with DAPI (Invitrogen) was added to cells. Cells were visualized by fluorescence microscopy using 600X magnification.
Anti aldolase
Manufactured by Abcam
Anti-aldolase is a laboratory reagent used to detect and measure the presence of the enzyme aldolase in biological samples. Aldolase is an enzyme involved in glycolysis, the metabolic pathway that converts glucose into energy. Anti-aldolase is a specific antibody that binds to and identifies aldolase, allowing for its quantification and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Lds buffer, by Thermo Fisher Scientific (1 mentions)
Novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Anti olfm4, by Cell Signaling Technology (1 mentions)
Donkey anti rat alexa fluor 555, by Abcam (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
3 protocols using anti aldolase
1
Immunofluorescence Visualization of Parasite Proteins
2
Immunoblotting Analysis of Parasite Proteins
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Parasites were lysed with 1% saponin for 5 min on ice. Parasite pellets were washed twice with ice-cold PBS and resuspended in 20 µl 1× lithium dodecyl sulfate (LDS) buffer (Life Technologies) per 1 ml culture at 2% hematocrit and 5% parasitemia. Equal parasite numbers were loaded per lane. After separation on Bis-Tris Novex gels (Invitrogen), proteins were transferred to a nitrocellulose membrane, blocked with a buffer containing 0.1% casein (Hammarsten; Affymetrix) and 0.2× PBS, and incubated with the corresponding antibodies diluted in 50% blocking buffer–50% Tris-buffered saline–Tween (TBST). Primary antibodies were used overnight at a 1:1,000 dilution, except antialdolase, which was used at 1:10,000, and anti-GFP, used at 1:20,000. Secondary antibodies were used at a 1:10,000 dilution for 1 h at room temperature. Blots were visualized using the LiCor double-color detection system and converted to grayscale images for the purpose of this publication. The following antibodies were used: anti-ATG8, from Josman LLC (see below); anti-aldolase (Abcam catalog no. ab207494); anti-ClpP, a gift from W. Houry (30 (link)); and anti-GFP (Clontech catalog no. 632381). Fluorophore-conjugated IRDye secondary antibodies were purchased from Fisher (LiCor).
3
Intestinal Organoid Immunofluorescence Imaging
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Immunofluorescence of intestinal organoids was performed as previously described70 (link). DAPI was combined with appropriate Alexa Fluor labeled secondary antibodies: Chicken anti-Rabbit Alexa Fluor 647 (ThermoFisher Scientific #A21443) 1:500; Donkey Anti-Rat Alexa Fluor 555 (Abcam #ab150154) 1:500; Donkey anti-Goat Alexa Fluor Plus 555 (ThermoFisher Scientific #A32816) 1:500. Images were collected on an inverted Leica TCS SP8 confocal microscope (Mannheim, Germany) in 12-bit with 25Xwater immersion objective (HC FLUOTAR L N.A. 0.95 W VISIR 0.17 FWD 2.4 mm). Imaris software (version 9.3.1, Oxford Instruments) was used for 3D reconstruction of images. The organoids were incubated with the following primary antibodies overnight: anti-Ly-6A (Sca1) (Biolegend #108101) 1:200, anti-Olfm4 (Cell Signaling #39141) 1:100, anti-Aldolase (Abcam #ab75751) 1:300, anti-Lysozyme (Agilent #A0099) 1:400, and anti-Mouse TROP-2 Antibody (R&D systems #AF1122) 1:50. Alexa Fluor labeled secondary antibodies (ThermoFisher Scientific #A22287) were combined with DAPI.
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