Spleen sections were analyzed using anti-CD11c, anti-ER-TR7 and anti-MADCAM-1 antibodies. Sections were fixed for 10 min with ice-cold acetone (CD11c, MADCAM-1) or for 15 min with 4% PFA (ER-TR7). Sections were rinsed with PBS and blocked for 30 min with 10% normal goat serum (in PBS). Primary antibodies were diluted in PBS and incubated for 1 h at room temperature. The sections were rinsed with PBS twice for 10 min. Secondary antibodies were diluted in PBS-Tween 0.1% supplemented with 2% normal goat serum, and incubated for 1 h at room temperature. Sections were rinsed with PBS twice for 10 min. Sections were covered with fluorescence mounting medium. Pictures were taken with a Zeiss Axioskop 2 plus fluorescent microscope. Exposure time of images was 2,000 ms. Hamster anti-mouse CD11c mAB (1∶100; BD, 550283, Franklin Lakes, NJ, USA) was used with the secondary AB goat anti-hamster AlexaFluor546 (1∶500; Invitrogen Ltd., Paisley, GB). Rat anti-mouse ER-TR7 mAB (1∶33; Abcam, ab51824, Cambridge, GB) and Rat anti-mouse MADCAM-1 mAB (1∶33; eBioscience, Inc., 14-5997-85, San Diego, CA, USA) were used with the secondary AB goat anti-rat AlexaFluor555 (1∶500; Invitrogen Ltd., Paisley, GB).
Axioskop 2 plus fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The Axioskop 2 plus is a fluorescent microscope designed for high-performance imaging. It features a high-intensity illumination system for fluorescence applications. The microscope is equipped with advanced optics to deliver clear, detailed images.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Axiovision software, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Axiocam hrm ccd camera, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Plan neofluar 100 1.30 oil objective, by Zeiss (1 mentions)
Illustrator cc, by Adobe (1 mentions)
Sodium citrate buffer, by Merck Group (1 mentions)
Terminal transferase, by Roche (1 mentions)
Biotinylated dutp, by Roche (1 mentions)
3 3 diaminobenzidine chromogen, by Merck Group (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Depex mounting medium, by Merck Group (1 mentions)
Terminal deoxy nucleotidyl transferase buffer, by Roche (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Pkh67 green fluorescent cell linker kit, by Merck Group (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin fungizone, by Lonza (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
7 protocols using axioskop 2 plus fluorescent microscope
1
Spleen Immunofluorescence Analysis
2
Multicolor Immunofluorescence Imaging of Splenocytes
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For both experiments, three- to four-color immunofluorescent staining was performed as precisely described in supplemental online materials for either phenotype (TCRαβ, etc.) of proliferating cells (EdU), nuclear transcription factor (Foxp3), tissue frameworks (type IV collagen), and DCs (CD103, etc.) in the spleen cryosections. Multicolor images were captured using an Axioskop2 Plus fluorescent microscope (Carl Zeiss, Jena, Germany) with an AxioCam MRm camera and AxioVision software (Carl Zeiss). Filters used were Filter Set 49 for Alexa-350, 17 for Alexa-488, 32 for Alexa-647 or -680 (Carl Zeiss), and XF407 for Alexa-594 (Omega Optical, Brattleboro, VT, USA), respectively. This filter combination had negligible crossing over of emitted lights between filters. We assigned pseudocolors to each channel to make merged images more comprehensible by maximizing contrast using AxioVision software (Carl Zeiss).
3
Imaging Barrier Cell Phenotypes
4
Immunostaining of Mitotic Spindle Proteins
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Cells adherent to Lab-Tek chamber slides (177,380) were rinsed in PBS. The cells were treated with BRB80 buffer for 5 min and then fixed for 10 min with 3.7% Formaldehyde in BRB80 buffer. After washing the cells in PBS for 5 min, they were treated with PBST (PBS+0.1% Triton X-100) for 15 min. Finally, the cells were blocked for 20 min in PBST containing 3% BSA. Both control and Tum/RacGAP dsRNA-treated cells were stained with anti-Tubulin diluted 1:200 (S9026 Sigma T clone DM1α), anti-Pav/kinesin-6 diluted 1:200 (kindly provided by D. Glover) or anti-Feo diluted 1:100 (Feo, kindly provided by M. Gatti) antibodies. Immunostained preparations were analysed with a Zeiss Axioskop 2 plus fluorescent microscope equipped with an AxioCam HRm CCD camera and images were acquired with a Plan-NEOFLUAR × 100 1.30 oil objective and Axiovision 4.6.3 software (Zeiss). Photoshop CC and Illustrator CC (Adobe) software were used to prepare the images. To confirm that the Pav/kinesin-6 signal observed in control and Tum/RacGAP RNAi-treated cells was specific to Pav/kinesin-6 primary antibody and not the result of non-specific binding or auto-fluorescence of the secondary antibody, we stained the cells with the Alexa Fluor 594 goat anti-rabbit secondary antibody alone.
5
Quantification of Cell Apoptosis by TUNEL
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Cell apoptosis was evaluated by terminal deoxy-nucleotidyl transferase dUTP nick-end labeling (TUNEL), as previously described [57 (link)]. Briefly, permeabilized cells were incubated in terminal deoxy-nucleotidyl transferase buffer (0.25 U/μl terminal transferase, 6 μM biotinylated dUTP, pH 7.5; all from Roche Farma, S.A, Madrid, Spain) for 1 hour 30 minutes at 37°C in a humidified chamber. The enzymatic reaction was stopped by 15 minutes of incubation in 300 mM NaCl (Sigma-Aldrich) and 30 mM sodium citrate buffer (Sigma-Aldrich). Following an additional rinse in PBS, cultures were incubated for 30 minutes at room temperature with the avidin-biotin-peroxidase complex (1:100; Vector Laboratories, Burlingame, CA). Peroxidase activity was revealed by the 3,3′-diaminobenzidine chromogen (0.025%; Sigma-Aldrich) intensified with 0.08% NiCl2 in 30 mM Tris-HCl (pH 7.6) buffer containing 0.003% H2O2. The cell preparations were then dehydrated in ethanol (70%, 2 minutes; 80%, 2 minutes; 90%, 2 minutes; 95%, 2 minutes; 100%, 2 minutes), cleared in xylene (3 minutes), and mounted using DEPEX mounting medium (Sigma-Aldrich). Photomicrographs of TUNEL were recorded using a digital camera (Axiocam HRC; Carl Zeiss) adapted to an Axioskop 2 Plus fluorescent microscope (Carl Zeiss). For each well, 8 random fields were photographed and positive cells were scored.
6
Exosome Uptake by Oligodendrocyte Progenitor Cells
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To confirm the uptake of exosomes into OPCs, exosomes were labeled using a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s protocol with minor modifications as previously described [42 (link)]. Three hundred microliters of each exosome suspension was mixed with 100 μL of Diluent C. Stain solution was prepared by adding 1.4 μL of PKH67 to another 300 μL of Diluent C. The exosome solution and stain solution were mixed, incubated at room temperature for four minutes, and the labeling reaction was stopped by adding 700 μL of FBS to the mixed solution. Labeled exosomes were added to OPCs and incubated at 37 °C for 24 hours. After the incubation, cells were fixed with 2% PFA solution. OPCs were immunostained using an anti-CD68 antibody (KP1, Dako, CA, USA) labeled with Alexa Fluor 594 goat anti-mouse IgG (A11005, Invitrogen) and observed using an Axioskop 2 plus fluorescent microscope (Carl Zeiss, Jena, Germany).
7
Culturing DRG Neurons with MSC Conditioned Media
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Dorsal root ganglia (DRGs) were dissected from adult male Wistar rats (3 months). The neurons were dissociated with 0.2% collagenase and 0.1% trypsin and then centrifuged through 15% bovine serum albumin (BSA) in DMEM (Thermo Fisher Scientific Inc.). DRGs were cultured in 24-well plates on coverslips coated with poly-D-lysine (20 µg/ml) and laminin (1 µg/ml) in DMEM, supplemented with penicillin–streptomycin–fungizone (1%, Lonza) and mitomycin C (0.25 µg/ml) mixed in 1:1 ratio with CM (prepared in the absence of PL) or α-MEM (N = 3 in duplicates for each of the MSC source). After 24 hrs of culture, the cells were fixed with 4% paraformaldehyde in PBS and stained with anti-beta III tubulin (Abcam) and goat anti-mouse IgG Alexa Fluor® 488 (Life Technologies). Images were acquired on Zeiss Axioskop 2 Plus fluorescent microscope. An analysis of the percentage of beta III tubulin positive area was calculated using ImageJ software 1.8.0_112 and related to the positive area of DRGs cultured in the control medium (α-MEM without supplements).
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