To evaluate lesion size and spared tissue area, tissue was collected for immunohistochemistry (n = 7 male and female rats) at 7 dpi. Rats received an intraperitoneal pentobarbital overdose and were transcardially perfused with 0.9% saline, then 4% paraformaldehyde. Spinal cords were suspended in paraformaldehyde overnight, cyroprotected in 30% sucrose, and cryosectioned (16 μm; Gaudet et al., 2015 (link)). For immunohistochemistry, slides were incubated with 10% normal donkey serum (1 h), then with primary antibodies (overnight; mouse anti-glial fibrillary acidic protein (GFAP; 1:100; catalog #0869110, MP-Biomedicals) and rabbit anti-Iba1 (1:1000; catalog #019-19741, Wako Chemicals), then with secondary antibodies (2 h; Alexa Fluor-488 donkey anti-mouse (A-21202) and Alexa Fluor-546 donkey anti-rabbit (A-10040); both 1:500; Thermo Fisher Scientific) and DAPI (nuclear stain; catalog #D1306, Thermo Fisher Scientific). Images were captured on an Olympus IX81 Microscope and analyzed using Fiji (Schindelin et al., 2012 (link)).
Alexa fluor 488 donkey anti mouse a 21202
Manufactured by Thermo Fisher Scientific
Alexa Fluor-488 donkey anti-mouse (A-21202) is a fluorescently labeled secondary antibody used for detecting and visualizing mouse primary antibodies in various immunoassays and microscopy applications. The Alexa Fluor-488 dye provides bright green fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Alexafluor 546 donkey anti rabbit a10040, by Thermo Fisher Scientific (2 mentions)
Catalog 019 19741, by Fujifilm (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Sp5 microscope, by Leica (1 mentions)
Lsm 700, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm confocal laser scanning microscope, by Zeiss (1 mentions)
4 protocols using alexa fluor 488 donkey anti mouse a 21202
1
Spinal Cord Injury Immunohistochemistry
2
Immunofluorescence Staining of A549 Cells
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A549 cells were seeded onto glass coverslips (1 × 105 per 24 wells) 1 day prior to infection at an MOI of 1. The cells were washed with PBS at 12 hpi and fixed overnight in PBS–4% paraformaldehyde (PFA) at 4°C. The coverslips were then washed with PBS, and the cells were permeabilized with PBS–0.1% Triton X-100, washed again, and blocked in PBS–1% FCS. Staining with primary antibodies diluted in blocking buffer (IRF3 FL-425, 1:200; SFSV, 1:2,500) was performed for 1 h in a humid chamber. Afterward, the coverslips were washed with PBS and incubated with secondary antibodies (Alexa Fluor 488 donkey anti-mouse [A21202] and Alexa Fluor 647 donkey anti-rabbit [A31573]; Thermo Fisher Scientific) (both 1:500) and 4′,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml) for 45 min in a humid chamber. Samples were washed again in PBS, rinsed in demineralized water, and mounted on microscopic slides using FluorSave reagent (Calbiochem). Confocal microscopy was performed using a Leica SP5 microscope and the accompanying software.
3
Immunofluorescence Analysis of CRC Cells
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Primary CRC cells were seeded and grown in 12-well cultivation chambers with removable microscopy glass slides (ibidi, Martinsried, Germany); cancer spheroids obtained by the hanging drop assay were also sedi-mented in the same chamber. Immunofluorescence analyses were performed as previously described by Di Maio et al (31 (link)). Briefly, following fixation in 4% paraformaldehyde in PBS for 10 min, the cells were permeabilized in 0.1% Triton X-100 in PBS for 30-120 min, and then blocked in 10% bovine serum albumin for 30 min. The cells were incubated with primary antibodies (Table I ) overnight, and then with secondary antibodies (Alexa Fluor 546 donkey anti-rabbit, A10040; Alexa Fluor 488 donkey anti-mouse, A21202; Thermo Fisher Scientific) for 1 h, and then with DAPI (Sigma-Aldrich) for 30 min at room temperature to label the nuclei. Negative controls without primary antibodies were also included, and these exhibited no staining. Following the indicated treatments, coverslips were mounted on glass slides and examined under a fluorescence confocal microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany).
4
Double Immunofluorescence Staining of Ubiquitin Linkages
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Double immunofluorescence staining was carried out with pairs of primary antibodies as follows: K48-Ub (clone D9D5) and L-Ub (LUB6), K48-Ub (clone D9D5) and K63-Ub (clone HWA4C4), L-Ub (LUB6) and K63-Ub (clone D7A11), L-Ub (LUB6) and HOIP (ab187976), L-Ub (LUB6) and SHARPIN (HPA044453), and L-Ub (LUB6) and optineurin (No. 100000). Primary antibodies were detected with Alexa Fluor 488-(donkey antimouse, A21202, Thermo Fisher Scientific, Waltham, MA; 1:150) and Alexa Fluor 647-(donkey antirabbit, A-31573, Thermo Fisher Scientific; 1:150) conjugated secondary antibodies. To suppress the autofluorescence of tissue, we processed sections with Sudan Black B. We observed sections with an LSM confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
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