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).
A21208
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A21208 is a laboratory instrument designed for general use in scientific research and analysis. It is a specialized piece of equipment that performs a core function within the laboratory setting. The detailed specifications and intended applications of this product are not included in this response, as an unbiased and factual description has been requested.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
A21202, by Thermo Fisher Scientific (5 mentions)
A31572, by Thermo Fisher Scientific (4 mentions)
A31571, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
A21206, by Thermo Fisher Scientific (4 mentions)
A21209, by Thermo Fisher Scientific (3 mentions)
A31573, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
A21432, by Thermo Fisher Scientific (3 mentions)
Dp controller software, by Olympus (2 mentions)
Dp30bw ccd camera, by Olympus (2 mentions)
A11055, by Thermo Fisher Scientific (2 mentions)
H 1200, by Vector Laboratories (2 mentions)
A32754, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
A31570, by Thermo Fisher Scientific (2 mentions)
A 11057, by Thermo Fisher Scientific (2 mentions)
A21207, by Thermo Fisher Scientific (2 mentions)
44 protocols using a21208
1
Immunofluorescence Staining of Frozen Tissue
2
Quantifying Neurogenesis in Fetal Mice
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Pregnant mice (E14.5) were injected intraperitoneally with BrdU at 0.1 mg/kg of body weight and euthanized at E18.5. Brains were fixed with 4% PFA solution in PBS for 3 h at 4 ℃ and cryopreserved in 30% sucrose in PBS. Brains were embedded in OCT and stored at − 80 ℃ until further use. The cryostat sections were cut at 12 μm and adhered onto glass slides. Then, the sections were washed with PBS and incubated with 10 mM citric acid (pH 6.0) for 30 min at 80 °C for antigen retrieval. The sections were incubated with 0.1 N HCl for 1 h at 37 °C. After incubation and brief washing with PBS, the sections were incubated with 0.5% Triton X-100 in PBS for 15 min at room temperature, and then incubated with blocking buffer (10% horse serum in TBST) for 30 min at room temperature. Further, the sections were incubated at 4 °C overnight with primary antibodies against BrdU (347,580, BD Bioscience; 1/100) and cleaved caspase3 (9661, Cell Signaling; 1/200), washed twice with TBST for 10 min, and incubated with the appropriate secondary antibodies conjugated with Alexa 488 or 546 (A11001, A21208, Invitrogen; 1/300) for 1 h at room temperature. Fluorescence microscopy was performed on a BX51 microscope equipped with a DP30BW CCD camera (Olympus) using 10× and 20× objective lenses. Images were processed using the DP controller software (Olympus).
3
Immunohistochemistry of Neurotransmitter Markers
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The primary antibodies used for immunohistochemistry were as follows: mouse anti-PV (1:1,000, 235, Swant), rabbit anti-SST (1:2,000, T-4103, Peninsula), mouse anti-RELN (1:1,000, MAB5364, Millipore), rabbit anti-VIP (1:500, 20077, Immunostar), goat anti-SATB1 (1:100, sc5889, Santa Cruz), anti-RFP (1:500, 600–401-379, Rockland), and rat anti-BrdU (1:500, OBT0030G, Accurate Chemicals). The secondary antibodies used for immunohistochemistry, all raised in donkey and used at 1:2,000 dilution, were as follows: anti-mouse Alexa Fluor 488 (A21202, Invitrogen), anti-mouse Alexa Fluor 555 (A31571, Invitrogen), anti-rabbit Alexa Fluor 555 (A31572, Invitrogen), anti-goat Alexa Fluor 488 (A11055, Invitrogen), anti-rat Alexa Fluor 647 (712–606-153, Jackson Immunoresearch), and anti-rat Alexa Fluor 488 (A21208, Invitrogen).
4
Multicolor Immunofluorescence Staining of Lung Immune Cells
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CD45+CD3+CD4+CD25+Foxp3+ Tregs, CD45+CD11b+CD64+ macrophages, and CD45+CD11b+CD64-CD11c+MHC II+ dendritic cells were sorted from the lung tissues. Cells were firstly centrifuged in a Cytospin centrifuge (800 rpm, 5 min, Thermo Shandon Cytospin 3) onto Cytospin slides which were air-dried. Fixed cell-slides were then coated in 4% paraformaldehyde (PFA) for no more than 10 min and carefully washed 5 min with sterile PBS three times. After fixation, coated-cells were subjected to a permeabilization with 0.1% Triton X-100 in PBS for no more than 10 min, washed with sterile PBS for 5 min, then embedded with blocking solution for 1 h at room temperature. The plants were then covered with Foxp3 (13-5773-82, eBioscience), CD11c (ab33483, Abcam), CD68 (ab213363, Abcam), p35 (ab131039, Abcam), and EBI3 (sc-166158, Santa Cruz Biotechnology), followed by embedding with secondary antibody (A21208, A32727 and A21094, Invitrogen) for 1 h and incorporated with 4',6-diamidino-2-phenylindole (DAPI) for 5 min.
5
Immunofluorescence Staining of VDR and Collagen I
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HSFs were fixed in freshly prepared 4% paraformaldehyde for 30 minutes at 4°C and washed three times in PBS for 5 minutes with a 0.1 M PBS blocking solution containing 6% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100 that was applied for 2 hours at room temperature to block nonspecific binding. Primary antibodies against VDR (1:100, 12550; Cell Signaling Technology, Danvers, MA, USA) and Collagen I (1:400, ab88147; Abcam, Cambridge, MA, USA) were diluted in the dilution buffer (containing 3% normal donkey serum, 0.5% bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PBS), which covered the sections during overnight incubation at 4°C. After washing the sections three times with PBS for 5 minutes each, they were incubated with secondary antibodies for 2 hours at room temperature with either donkey anti-rat IgG (H+L) conjugated to Alexa Fluor 488 (1:400, A-21208; Invitrogen) or donkey anti-rabbit IgG (H+L) conjugated to Alexa Fluor 555 (1:400, A-31572; Invitrogen). The secondary antibodies were diluted in the same solution as the primary antibody. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). The images were captured with a Zeiss LSM 880 confocal microscope (ZEISS, Göttingen, Germany) under a ×20 magnification objective.
6
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).
7
Immunostaining of Differentiated Human iPSCs
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The differentiated human iPSCs were fixed on glass coverslips (VWR, Denmark) in 4% PFA for 15 min at RT and stored at 4°C until use. The cells were washed in PBS and permeabilized in 0.1% Triton X-100 in PBS for 1 h, washed in PBS and antigen retrieval was performed by immersing cells into boiling one x citrate buffer 3 times for 5 min each. Cells were treated with blocking buffer (5% NDS in PBS) for 1 h at RT. Cells were incubated overnight at four°C in primary antibodies targeted against BCL11B (1:750, Abcam, ab18465), Reelin (1:50, Santa-Cruz, sc-25346), SATB2 (1:800, Abcam ab34735), Nestin (1:500, Millipore, Abd69) and MAP2ab (1:200, Sigma, M1406) diluted in blocking buffer. The cells were washed 3 × 5 min in PBS and incubated with secondary antibodies conjugated with Alexa fluorophores (1:200, Invitrogen, A10036, A21208, and A21448) diluted in blocking buffer for 1 h at RT. The cells were then washed three times for 5 min in PBS and counterstained with Hoechst 33342 (1 μg/ml in PBS) for 10 min. The cells were washed in PBS and mounted onto glass microscope slides using buffered glycerol mounting media 90% glycerol (Sigma-Aldrich), 20 mM Tris pH 8.0, 0.5% N-propyl gallate (Sigma-Aldrich).
8
Immunofluorescence Staining of Neural Markers
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The frozen sections (n = 3 per group) were first permeabilized using 0.1% Triton X-100 for 10 min followed by blocking with 5% donkey serum in PBS for 30 min. The sections were then incubated with primary antibodies overnight at 4 °C, subsequently rinsed with PBS, and incubated with secondary antibodies at RT for 1 h. After rinsing with PBS for 3 times, the slides were mounted using Vectashield mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). The slides were subsequently imaged under immunofluorescence microscope (Axio Scope.A1, Zeiss, Oberkochen, Germany).
The primary antibodies used in this study included mouse anti-NeuN (1:400, ab104224, Abcam, Cambridge, England), mouse anti-GFAP (1:100, sc-33673, Santa Cruz Biotechnology), rat anti-CD68 (1:200, MCA1957, Bio-Rad Laboratories, Hercules, USA), sheep anti-PGRN (1:200, AF2557, R&D Systems, Minneapolis, USA), and rabbit anti-iNOS (1:200, PA1-036, Invitrogen, Carlsbad, USA). The secondary antibodies used in this study included donkey anti-mouse Alexa Fluor 488 (1:200, A-11017, Invitrogen, Carlsbad, USA), donkey anti-rat Alexa Fluor 488 (1:200, A-21208, Invitrogen, Carlsbad, USA), donkey anti-rabbit Cy3 (1:200, AP182C, Sigma-Aldrich, Darmstadt, Germany), and donkey anti-sheep Alexa Fluor 647 (1:200, A-21448, Invitrogen, Carlsbad, USA)
The primary antibodies used in this study included mouse anti-NeuN (1:400, ab104224, Abcam, Cambridge, England), mouse anti-GFAP (1:100, sc-33673, Santa Cruz Biotechnology), rat anti-CD68 (1:200, MCA1957, Bio-Rad Laboratories, Hercules, USA), sheep anti-PGRN (1:200, AF2557, R&D Systems, Minneapolis, USA), and rabbit anti-iNOS (1:200, PA1-036, Invitrogen, Carlsbad, USA). The secondary antibodies used in this study included donkey anti-mouse Alexa Fluor 488 (1:200, A-11017, Invitrogen, Carlsbad, USA), donkey anti-rat Alexa Fluor 488 (1:200, A-21208, Invitrogen, Carlsbad, USA), donkey anti-rabbit Cy3 (1:200, AP182C, Sigma-Aldrich, Darmstadt, Germany), and donkey anti-sheep Alexa Fluor 647 (1:200, A-21448, Invitrogen, Carlsbad, USA)
9
Multicolor Immunofluorescence for Ki67, CD45, and Endomucin
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Slides containing cytospin-isolated cells were fixed and permeabilized with 3.7% formaldehyde and 0.1% triton for 20 min at room temperature (RT). After blocking with 5% BSA in PBS for 1 h, slides were incubated with anti-Ki67 (1:200, thermofisher-RM-9106), CD45 (1:200, Invitrogen-16-0451), and endomucin antibodies (1:200, R&D-AF4666) overnight at 4 °C. Slides were washed with PBST buffer followed by incubation with donkey anti-rat (1:500, Invitrogen A-21208), donkey anti-goat secondary (1:500, Invitrogen A-11055) or antibodies conjugated with Alexa Fluor 488 or donkey anti-rabbit IgG conjugated with Alexa Fluor 647 (1:500, Invitrogen A31573) in a dark chamber for 1 h at RT. Nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI). Images were captured using Nikon Fluorescent microscope.
10
Immunohistochemistry of Mouse Whole Eyes
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Mouse whole eyes were sectioned (9 μm in thickness) and fixed in 4% paraformaldehyde for 20 min. The slides were incubated in 0.2% Triton X-100 for 20 min and then 2% BSA for 1 h at room temperature. Slides were incubated with anti-aSMA (1:200 dilution, 14-6496-82, eBioscience, San Diego, CA, USA) or anti-CD45 (1:100 dilution, 103101, BioLegend, San Diego, CA, USA) antibodies at 4 °C overnight, followed by Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:500 dilution, A-21202, Invitrogen, Carlsbad, CA, USA) or donkey anti-rat secondary antibody (1:500 dilution, A-21208, Invitrogen) for 1 h at room temperature. The slides were mounted with DAPI mounting medium (H-1200, Vector Lab, Burlingame, CA, USA) and photographed under a confocal laser scanning microscope (SP8, Leica, Wetzlar, Germany).
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