The frozen sections were fixed with 4% paraformaldehyde (Sigma #P6148) and permeabilized with 0.25% Triton X-100 (Fisher Scientific #BP151–100). After a wash with PBS/Tween-20 (Fisher Scientific #BP337–100), slides were treated with 3 M hydrochloric acid (Fisher Scientific #A144S-500) for 10 min to open the nucleus structure if needed. Slides were blocked with 10% donkey serum (Sigma #D9663) in 2% BSA (Sigma) in PBS/Tw-20 for 1 h at room temperature. Afterwards, slides were probed with primary antibodies (CD31 1:25, BD #550274; BrdU-APC 1:50, BD; Alexa Fluor 488 Mouse anti-BrdU 1:10, BD #558599; Flag M2 1:500, Sigma #F1804) and incubated overnight at 4 °C. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen #A-21208; AF594 donkey anti-mouse 1:300, Invitrogen #A-21203; AF594 donkey anti-rat 1:300, Invitrogen #A-21209). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).
A21203
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A21203 is a laboratory equipment product offered by Thermo Fisher Scientific. It serves as a core function in various laboratory applications, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
A21206, by Thermo Fisher Scientific (40 mentions)
A21207, by Thermo Fisher Scientific (22 mentions)
A21202, by Thermo Fisher Scientific (20 mentions)
A11055, by Thermo Fisher Scientific (11 mentions)
A11058, by Thermo Fisher Scientific (9 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (6 mentions)
A31573, by Thermo Fisher Scientific (6 mentions)
A21209, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
A31571, by Thermo Fisher Scientific (4 mentions)
A11008, by Thermo Fisher Scientific (4 mentions)
Ab11959, by Abcam (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
A10036, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
A21447, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
79 protocols using a21203
1
Immunofluorescence Staining of Frozen Tissue
2
Immunohistochemical Staining of FFPE Tissues
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Formalin-fixed paraffin-embedded sections were deparaffinized with Histoclear (National Diagnostics) and rehydrated in a series of EtOH solutions. Antigen retrieval was performed in a microwave for 20 min in a 10 mM Citrate, 0.05% Tween-20, pH 6.0 solution. Sections were blocked in 3% bovine serum albumin (BSA) (Sigma) for 1 h, followed by overnight incubation in the primary antibody diluted in 3% BSA (Sigma, A2153). For immunofluorescence, detection and labeling were performed with secondary antibodies conjugated to Alexa-Fluor-488 or Alexa-Fluor-594 fluorophores (Invitrogen, A21203, A21206, or A21209) at a dilution of 1:250 in 3% BSA. For peroxidase staining, sections were washed and incubated in Biotinylated secondary antibodies (Goat Anti-Rabbit IgG Biotinylated, Vector Biolabs, BA-1000 at 1:200 dilution in 3% BSA; Goat Anti-Rat IgG Biotinylated, Vector Biolabs, BA-9400, 1:200 dilution in 3% BSA; Vector Mouse on Mouse Immunodetection Kit, Vector Biolabs, FMK-220 following manufacturer’s conditions), followed by avidin/biotin complex formation (Vectastain ABC, PK-6100, Vector Biolabs). Sections were then incubated with DAB peroxidase (horseradish peroxidase) substrate (Vector labs, SK-4100) and counterstained with hematoxylin. Immunofluorescence images were obtained using a Zeiss LSM 880 confocal microscope. All antibodies and dilutions are listed in Supplementary Table 3 .
3
Brain Barrier Integrity Evaluation
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After terminal anaesthesia, mice were perfused transcardially with saline followed by ice-cold 4% paraformaldehyde. Brains and spleens were post-fixed for 24 h, cryoprotected in 10% sucrose/PBS and sectioned (25 μm diameter) on a sledge microtome. Detection of IgG leakage to the brain parenchyma by immunostaining (Vector, NBA-2000, horse anti-mouse biotynilated IgG 1:500) was used to assess BBB permeability on brain sections as described earlier58 (link). Immunofluorescence was performed on free-floating brain sections using combinations of rabbit anti-Iba1 (WAKO, 019-19741, 1:500), rat anti-CD45 (AbDSerotec, MCA1388, 1:250), mouse anti-GFAP (Sigma, G3893, 1:500), rabbit anti-Claudin-5 (Invitrogen, 34-1600, 1:500) and goat anti-PDGFRβ (R&D Systems, AF1042, 1:500) antibodies. Sections were incubated in a primary antibody cocktail overnight followed by adequate fluorochrome (Invitrogen, A21206, donkey anti-rabbit Alexa 488 1:500; Invitrogen, A21203, donkey-anti-mouse Alexa 594 1:500)-conjugated antibodies. Biotinylated tomato lectin (Sigma-Aldrich, L0651-1 mg, 1:100) was used to visualize blood vessels, followed by streptavidin Alexa 350 conjugate (Invitrogen, S11249, 1:200). Images were captured using a Zeiss Axiovert 200M microscope with Axiovision 4.8 software.
4
Antibody Panel for Cytoskeletal Proteins
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The following antibodies were used: acetylated α-tubulin (T6793, clone 6-11B-1; Sigma) and βIII-tubulin (Tuj1, T8578, clone 2G10; Sigma), MBP (ab7349; Abcam), HA (11867431001, clone 3F10; Roche), contactin 1 (sc-20296; Santa Cruz Biotechnology). Rabbit polyclonals anti-Gal-4 and anti-Gal-3 (in-house production) were purified by affinity chromatography-based depletion, using the respective galectin as bead-immobilized ligand, and further tested by means of ELISA screening and Western blot against other commonly expressed members of this family, i.e. galectins-1, -2, -7, -8, and -9, to exclude cross-reactivity51 (link). F-actin was detected with TRITC- or FITC-conjugated phalloidin (P1951 and p5282, respectively, Sigma). Appropriate fluorescent anti-species secondary antibodies were from Invitrogen (A21202; A21203; A21206; A21207; A11055; A11058; A31571; A31573).
5
Immunofluorescence Staining of Cardiac Tissue
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Deparaffinizing, antigen retrieval, and blocking of non-specific background were performed as described previously 22 (link) and sections were incubated with primary antibodies overnight at 4°C. After washing three times with PBS, and incubated with fluorescence-labeled secondary antibodies for 1 hour at room temperature in the dark. Slices were washed three times in PBS, and then counterstained with DAPI (Sigma-Aldrich, St Louis, MO, USA). The primary antibodies were as follows: anti-sarcomeric alpha actinin (1:200; ab9465, Abcam, UK), anti-phosphorylated Histone 3 (1:1000; 3377, Millipore, USA), anti-Ki67 (1:200; ab16667, Abcam, UK), anti-Aurkb (1:100; A5102, Sigma, USA), anti-CDH5 (1:100; ab33168, Abcam, UK) and anti-Vimentin (1:100; ab92547, Abcam, UK). The secondary antibodies were from Invitrogen: anti-rabbit Alexa Fluor 488-conjugated (1:400; A21206, Invitrogen, USA), anti-mouse Alexa Fluor 594-conjugated (1:400; A21203, Invitrogen, USA). Fluorescence was observed under a ZEISS LSM800 confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Germany). We used High Content Screening System (Opera Phenix) to count cell proliferation ratio.
6
Histological and Immunofluorescence Analysis of Epididymal Adipose Tissue
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Epididymal fat was harvested at indicated time points (six mice per group for each time point). The epididymal adipose tissues were fixed in formalin and embedded with paraffin. Sections were cut at 6 μm and stained with hematoxylin-eosin (HE) according to a standard protocol. The size of adipocytes was measured using digital image analysis. For immunofluorescence, the adipose tissues were fixed in 4% paraformaldehyde, dehydrated with 50% sucrose, and embedded in OCT (Sakura, Finetek, USA, 4583). Frozen sections (10 μm thick) (3 slides per mouse) were blocked in BSA and incubated overnight at 4 °C with primary antibodies against Fizz1 (Abcam, San Francisco, USA, ab39626) or iNOS (Abcam, ab15323), followed by incubation with Alexa Fluor 488/594-conjugated secondary antibodies (Invitrogen, A21203, A31556, A-32766) at room temperature for 2 h. Finally, 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, B2261) was added. Sections were observed using a confocal laser scanning microscope (Olympus FluoView 1000, Tokyo, Japan, TCSSP8). BMDMs were plated onto glass coverslips and fixed with 4% paraformaldehyde. The next steps were performed as described above.
7
Immunofluorescence Staining of Cell Markers
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Deparaffinization, antigen retrieval with 1 mM EDTA (pH 9.0) in boiling water, and blocking of nonspecific binding sites were performed. The sections were then incubated with primary antibodies overnight at 4°C, washed three times with PBS, and incubated with fluorescence-labeled secondary antibodies for 1 h at 25°C in the dark. The slides were washed three times in PBS, counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA), and mounted with VECTASHIELD (Vector Labs, CA, USA). The primary antibodies used were as follows: anti-phospho Histone H3 Ser10 (Millipore #06-570, 1 : 100), anti-Ki67 (Abcam, ab16667, 1 : 200), anti-Aurora B (1 : 100; ab2254, Abcam), and anti-Sarcomeric Alpha Actinin (Abcam, ab9465, 1 : 500). The anti-rabbit Alexa Fluor 488-conjugated (1 : 500; A-21206) and anti-mouse Alexa Fluor 594-conjugated (1 : 500; A-21203) secondary antibodies were from Invitrogen. Fluorescence was observed under a ZEISS LSM800 confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Germany).
8
Immunostaining for SCI Markers
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Immunostainings for RIP3 and pMLKL were performed using the spinal cord sections obtained 3 days, and those for NeuN and RT97 were performed using sections obtained 42 days after SCI. The sections were washed in PBS for 15 min, after which they were washed with PBS containing 0.3% Tween for 10 min and blocked with 3% milk and 5% FBS in 0.01 M PBS for 2 h. The sections were incubated with rabbit anti-RIPK3 antibody (1:100; PRS2283, Sigma-Aldrich), rabbit anti-phospho-MLKL antibody (1:150; ab196436, Abcam, Cambridge, UK), mouse anti-NeuN antibody (1:100; MAB377, Millipore, Burlington, MA, USA) or mouse anti-RT97 antibody (1:30; AB528399, DSHB, Iowa City, IA, USA) diluted in PBS overnight at 4 °C. After rinsing with PBS, the sections were incubated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (1:500; A11034, Invitrogen, Carlsbad, CA, USA), donkey anti-rabbit IgG Alexa Fluor 594 secondary antibody (1:500; A21207, Invitrogen), goat anti-mouse IgG Alexa Fluor 488 secondary antibody (1:500; A11001, Invitrogen), or donkey anti-mouse IgG Alexa Fluor 594 secondary antibody (1:500; A21203, Invitrogen) for 1 h at room temperature. The sections were mounted with Vectashield containing DAPI to label the nuclei (Vector Laboratories). The sections were stained at the same time in each experiment.
9
Immunofluorescence Staining of Pluripotent Markers
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Cells were fixed with 4% PFA, permeabilized using 0.2% Triton X-100, and treated with Ultra Vision block (ThermoFisher). Primary antibodies were diluted in 0.1% Tween-20 PBS and incubated either overnight at 6 °C with the given dilutions or 2 days in 6 °C with halved primary antibody amounts. Secondary antibody incubations were done in room temperature for 30 min in the presence of Hoechst33342 to stain the nuclei. Primary antibodies used were: LIN28A (1:250, D84C11 and D1A1A, Cell Signaling), NANOG (1:250, D73G4, Cell Signaling), OCT4 (1:500, sc-8628, Santa Cruz), SOX2 (1:250, D6D9, Cell Signaling), KLF4 (1:250, HPA002926, Sigma-Aldrich), C-MYC (1:250, D3N8F, Cell Signaling; 1:250, [Y69] ab32072, Abcam), TRA-1-60 (1:50, MA1-023, ThermoFisher), TRA-1-81 (1:100, MA1-024, ThermoFisher) TUBB3 (1:500, MAB1195, R&D Systems), AFP (1:400, A0008, Dako), SMA (1:200, A2547, Sigma), VIMENTIN (1:500, sc-5565, Santa Cruz). SOX17 (1:500, AF1924, R&D Systems), acetyl Histone 3 (1:500, ab47915, Abcam). Secondary antibodies used were: AlexaFluor 488: donkey anti-goat (1:500, A11055 and 11058; Invitrogen), donkey anti-mouse (1:500, A21202 and A21203; Invitrogen) and donkey anti-rabbit (1:500, A21206 and A21207; Invitrogen).
10
Immunofluorescence Staining of Tight Junctions
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Immunofluorescence staining was performed [43 (link)] to evaluate ZO-1 and occludin expression in the colon. Anti-TSLP (rabbit, 1:50; GTX85059, GeneTex, Irvine, CA, USA), anti-IL-6R (rabbit, 1:1000; Invitrogen, Waltham, MA, USA), anti-ZO-1 (rabbit, 1:500; 61–7300, Invitrogen, Waltham, MA, USA), and anti-occludin (mouse, 1:200; OC3F10, Invitrogen, Waltham, MA, USA) antibodies were diluted in 1× PBST supplemented with 0.3% BSA. The slides were wrapped in an aluminum foil to block light and stored at 4 °C for 72 h. Tissue sections were incubated for 1 h with a mixture of Alexa 488-conjugated donkey anti-mouse secondary antibody (1:1000; A21206, Invitrogen, Waltham, MA, USA) or Alexa 594-conjugated donkey anti-mouse secondary antibody (1:1000; A21203, Invitrogen, Waltham, MA, USA).
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