Primary antibodies were mouse anti-BKα (ab192759; Abcam), rabbit anti-BKβ4 (ab222083; Abcam), and rat anti-HA (11867423001; Roche Applied Science). Secondary antibodies were goat anti-rat conjugated to Alexa Fluor 647 (ab150159; Abcam), goat anti-rabbit conjugated to Alexa Fluor 647 (A21245) or Alexa Fluor 488 (A11008), and goat anti-mouse conjugated to Alexa Fluor 647 (A32728) or Alexa Fluor 488 (A11001), all from Invitrogen. Cells at a density of 4 × 105 per well were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde (Electron Microscopy Sciences, EM grade) in PBS for 10 min at room temperature and then reduced with 0.1% NaBH4 in PBS for 7 min to mitigate cell auto-fluorescence. Next, cells were washed three times with PBS (5 min per wash) and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. Subsequently, cells were blocked for 90 min with 10% normal goat serum and 0.05% Triton X-100 in PBS and incubated with primary antibodies for 1 h. Samples were washed five times with 1% normal goat serum and 0.05% Triton X-100 in PBS (10 min per wash), incubated with secondary antibodies for 1 h, and washed again. Cells were then fixed with 3% paraformaldehyde and 0.1% glutaraldehyde for 10 min, rinsed three times with PBS, and stored at 4°C until used.
A 21245
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A-21245 is a laboratory centrifuge designed for general-purpose sample separation. It features a compact benchtop design and accommodates a variety of rotor options to handle different sample volumes and tube sizes. The centrifuge provides precise speed and time controls to enable consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
A21236, by Thermo Fisher Scientific (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
A11001, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
A11029, by Thermo Fisher Scientific (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
A11039, by Thermo Fisher Scientific (3 mentions)
A31570, by Thermo Fisher Scientific (3 mentions)
A11006, by Thermo Fisher Scientific (3 mentions)
A11011, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
A21448, by Thermo Fisher Scientific (3 mentions)
Vectashield, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
A11017, by Thermo Fisher Scientific (2 mentions)
A32728, by Thermo Fisher Scientific (2 mentions)
Ab13970, by Abcam (2 mentions)
A11034, by Thermo Fisher Scientific (2 mentions)
A11004, by Thermo Fisher Scientific (2 mentions)
64 protocols using a 21245
1
Multicolor Immunofluorescence Staining Protocol
2
Immunostaining of Synaptic Proteins
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Cells stained against syntaxin-1 (1:500; 110011/110302; Synaptic Systems), SNAP-25 (1:1000; 111011; Synaptic Systems), or EGFP (1:1500; ab13970; Abcam) were isolated and prepared as described. Cells were fixed in 4% paraformaldehyde (PFA) for 15 min, washed, permeabilized in 0.1% Triton X-100 (T8787; Sigma-Aldrich), and blocked in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA; A4503; Sigma-Aldrich). The cells were incubated with primary antibodies overnight at 4°C, washed, and incubated with secondary antibodies (Alexa Fluor 488–conjugated goat anti-chicken [A11039; Molecular Probes], Alexa Fluor 546–conjugated goat anti-rabbit [A11810; Invitrogen], Alexa Fluor 647–conjugated goat anti-mouse [A21235; Molecular Probes], and Alexa Fluor 647–conjugated goat anti-rabbit [A21245; Invitrogen]) for 2 h at room temperature, washed, and mounted on a microscope slide. Finally, samples were mounted with Fluorsave (Dako). Micrographs were recorded at room temperature.
3
Immunofluorescent Staining of Cellular Proteins
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Cells were grown on glass cover slips. Cytoplasm was pre-extracted by placing cover slips on ice in pre-chilled 0.5% Triton X-100/PBS for 5 min. Immunofluorescent staining was carried out as published15 using the following antibodies: α-PML (Santa Cruz sc966 1:500), α-RPA (P-S33, Bethyl A300-246A 1:2,000), α-LANA (Millipore MABE1109, 1:250), α-mouse-Alexa647 (Invitrogen A21236 1:500), α-rat-Alexa488 (Invitrogen A1106 1:500), and α-rabbit-647 (Invitrogen A21245, 1:500). After staining, three washes with PBST were carried out followed by re-fixation in 2% Methanol-free Formaldehyde for 5 min. Telomere probe (TelC-Cy5, PANAGENE #F2001) was applied at a final concentration of 4 nM as described42 .
4
Immunohistochemical Profiling of Immune Cells
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Four-µm-thick tissue sections prepared from formalin-fixed and paraffin-embedded tissue samples were deparaffinized in xylene twice for 10 min each and rehydrated in a graded ethanol series for 10 min each. After blocking endogenous peroxidase by 3% hydrogen peroxide and antigen retrieval by 14 min of microwave heating in EDTA buffer (pH 7.4), the samples were incubated with primary antibodies against CD8 (1:400, ab199016, Abcam, Cambridge, UK), CD103 (1:200, ab129202, Abcam), and CD69 (1:200, bs-2499R, Bioss, Woburn, MA, USA) overnight at 4 °C, and then incubated with peroxidase-linked secondary antibody for 30 min at room temperature. The samples were immersed in 3, 3-diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin, followed by dehydration in a graded ethanol series for 10 min each. After mounting, the samples were subjected to microscopic observation. For fluorescence immunohistochemical staining for CD8 and CD103, Alexa 488 Anti-mouse (1:500, A11017, Invitrogen, Tokyo, Japan) or Alexa 647 Anti-Rabbit (1:500, A21245, Invitrogen) was used as the secondary antibody, and DAPI (D21490, FluoroPure™ grade, Invitrogen) was used for DNA staining.
5
Immunostaining of Drosophila Fat Body and Midgut
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Bisected third instar larvae were inverted and fixed with 3.7% paraformaldehyde in PBS overnight at 4 °C. Next, samples were rinsed twice and washed for 2 h in PBS, permeabilized for 15 min in PBTX-DOC (PBS with 0.1% Triton X-100 and 0.05% sodium deoxycholate) and blocked for 3 h in 3% goat serum in PBTX-DOC. Samples were then incubated overnight at 4 °C with primary antibodies in 1% goat serum in PBTX-DOC. After 3 × 30 min washes in PBTX-DOC, samples were incubated with secondary antibodies diluted 1:1,500 in 1% goat serum in PBTX-DOC for 4 h at room temperature. Finally, after 3 × 15 min washes in PBTX-DOC and 1 × 15 min in PBS, fat bodies and midguts were dissected and mounted in 50% glycerol/PBS with 0.2 μM DAPI. The following secondary antibodies were used: Alexa 488 anti-chicken, Alexa 488 anti-rabbit, Alexa 546 anti-rabbit, Alexa 568 anti-rat, Alexa 568 anti-mouse, Alexa 647 anti-rabbit (all used in 1:1,500, Invitrogen A11039, A11034, A11035, A11077, A1104, A21245, respectively).
6
Immunostaining of Circulating Tumor Cells
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Cells were immunostained with primary antibodies at room temperature for 30 min following fixation and permeabilization with 4% paraformaldehyde (PFA) (Cat No. 09154-85, Nacalai-Tesque, Kyoto, Japan) and 0.15% Triton X-100, (Cat No. 93343-100ML, Sigma) respectively. Primary antibodies used were anti-CD45 (Cat No.304002, 1:100 or 304032, 3:100; BioLegend, San Diego, CA), anti-CD45 conjugated with Brilliant Violet 421 (304032, 3:100; BioLegend), anti-EpCAM (ab7504, 1:100; Abcam), anti-CD133 (130-090-422, 1:20; Miltenyi Biotec), anti-vimentin (ab45939, 1:200; Abcam, Cambridge, UK), anti-pan-cytokeratin (628602, 1:100; BioLegend), anti-cytokeratin 19 (628502, 1:100; BioLegend), and anti-CEA (M707229, 1:50; Dako Corp., Carpenteria, CA). For signal amplification, secondary antibodies (A21422, A21235, A11046, A21245, 1:200; Invitrogen, Carlsbad, CA) or a labeling kit (Z25005, Invitrogen) were used. Nuclear DNA was stained with SYTOX Blue (Molecular Probes, Eugene, OR) to confirm the viability of the CTCs. Cell surface markers were immunostained before fixation.
7
Nestin-CFP Immunohistochemistry in Pons
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Nestin‐CFPnuc mice were euthanized at P3, brains were extracted and fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) for 24 h and cryopreserved in 30% sucrose in PBS for 24–48 h. After embedding in OCT compound, blocks were sectioned using a Cryostat (TN50, Tanner Scientific, Inc.) into 10 μm thick sagittal or oblique coronal sections to visualize the whole pons (Lindquist et al., 2016 (link)). Sections were rehydrated in PBS with 0.1% Triton X‐100 (PBS‐T). Antigen retrieval was performed using citrate buffer pH 6 for 20 min at 95°C. Sections were then permeabilized using PBS‐T with 0.3% Triton X‐100 and blocked with PBS‐T containing 5% normal donkey serum. Anti‐Olig2 (Millipore, #AB9610, 1:500), anti‐Sox2 (Abcam, ab79351, 1:200), and anti‐GFP (Nacalai, 4404‐84, 1:1000, to label CFP) primary antibodies were used. AlexaFluor donkey anti‐rabbit‐488 (Invitrogen, A21208, 1:400), donkey anti‐mouse‐594 (Invitrogen, A31570, 1:400) and goat anti‐rabbit‐647 (Invitrogen, A21245, 1:400) were used as secondary antibodies. Slides were mounted with Vectashield with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Vector Laboratories) and imaged using a Lionheart™ automated microscope (BioTek, Vermont). The percentages of Nestin‐CFP+, Olig2+, and double‐positive cells were quantified with Fiji software (NIH).
8
Antibody Profiling for Toxoplasma Infection
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Antibodies used in this study were rabbit pAb anti-p62 (#PM045, MBL; RRID:AB_1279301 ), mouse mAb anti-GRA2 (A1298, Biovision) mouse mAb anti-ubiquitin FK2 (PW8810, Enzo Lifesciences; RRID:AB_10541840 ), mouse mAb anti-SAG1 (home-made) and rat mAb anti-HA (11867423001, Sigma). Secondary antibodies used were Alexa Fluor 647-conjugated goat anti-rabbit (A-21245, Invitrogen; RRID:AB_141775 ), anti-rat (A-21247, Invitrogen; RRID:AB_141778 ) or anti-mouse (A-21236, Invitrogen; RRID:AB_141725 ), Alexa Fluor 488-conjugated goat anti-mouse (A-11001, Invitrogen; RRID:AB_2534069 ) and Alexa Fluor 568-conjugated goat anti-mouse (A-11004, Invitrogen; RRID:AB_141371 ).
9
ACE2 Expression in Stimulated HAECs
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HAECs were seeded into gelatin-coated 96-well plates and stimulated with 50 ng/mL TNFα or 10 ng/mL IL-1β for 5 h, 24 h, or 5 d. After the incubation, the cells were fixed with 4% PFA (Sigma-Aldrich) for 10 min. Membrane permeabilization was performed with 0.2% Triton-X 100 (Serva, Heidelberg, Germany) and blocking of unspecific binding sites with 3% goat serum (Abcam). The cells were stained with primary rabbit monoclonal antibody against ACE2 (1:100, #MA5-32307, Invitrogen) and goat anti-rabbit Alexa Fluor 647 (1:500, #A21245, Invitrogen) antibody. Nuclear counterstaining was performed by using 1 µg/mL Hoechst 33258 (Cayman Chemical, Ann Arbor, MI, USA). Fluorescent images were taken on an inverted Olympus IX71 microscope (Olympus, Shinjuku, Japan) with a 10× air objective using the 385 nm excitation line with a 447/60 blue emission filter (DAPI/Hoechst) and the 660 nm excitation line with a 692/40 Far red emission filter for ACE2. For quantification, background correction was performed, and ACE2 staining intensity was normalized to the DNA staining intensity.
10
Visualizing Osteogenic Differentiation of hMSCs
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