To analyze the tissue expression of the ARSA protein, transverse or sagittal cryostat sections of CNS and PNS organs obtained from minipigs were used. For immunofluorescent labeling, sections were blocked with 5% normal goat serum then incubated with a primary antibody (anti-ARSA, Cloud-Clone, Tokyo, Japan) and subsequently incubated with secondary antibodies (Alexa 546, Abcam, Cambridge, UK). Following successive washes in PBS, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (10 μg/mL in PBS, Sigma-Aldrich) to visualize nuclei. Sections were mounted with a medium (ImmunoHistoMount, Santa Cruz, CA, USA) and examined using an LSM 700 confocal scanning microscope (Carl Zeiss, Jena, Germany).
Alexa 546
Manufactured by Abcam
Sourced in United Kingdom
Alexa 546 is a fluorescent dye used for labeling and detection in various biological applications. It is a synthetic fluorophore that emits light in the red-orange region of the visible spectrum. Alexa 546 is commonly used for immunofluorescence, flow cytometry, and other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal scanning microscope, by Zeiss (1 mentions)
Immunohistomount, by Santa Cruz Biotechnology (1 mentions)
Cryotome, by Leica (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Myelin basic protein, by BioLegend (1 mentions)
Alexa 488, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Cd11b, by Bio-Rad (1 mentions)
Alexa 555, by Abcam (1 mentions)
Alexa 647, by Abcam (1 mentions)
3 protocols using alexa 546
1
Tissue Expression Analysis of ARSA Protein
2
Immunofluorescence in Acute Hippocampal Slices
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For immunofluorescence, animals were anesthetized with 4% isoflurane and acute hippocampal slices were prepared as described in the section on slice preparation. The slices were then placed in 4% paraformaldehyde for 1 h at room temperature. After washing with PBS, the slice was transferred to a 30% sucrose solution in PBS and left overnight at 4 °C. Sections (40 μm) were cut with a cryotome (Leica Biosystems, Wetzlar, Germany). The sections were then mounted and permeabilized with 0.3% Triton X-100 in PBS for 30 min, and nonspecific binding sites were blocked with 5% goat serum in PBS for two hours at room temperature. Sections were then incubated with primary antibody (anti-RGS14 1:200; Abcam, Waltham, MA, USA) overnight at 4 °C. After three washes with PBS, sections were incubated with a species-specific secondary antibody (goat anti-rabbit Alexa 546, 1:400; Abcam) in 5% goat serum for 1 h. After application of DAPI in 0.01 M PBS (100 ng/mL, 28718-90-3, Roche, Switzerland) for 5 min and three additional washes with PBS, the sections were mounted onto slides (Fluoromount Aqueous Mounting Medium, Sigma, Livonia, MI, USA), and fluorescence images were captured using a fluorescence microscope system.
3
Immunohistochemical Analysis of Nerve and Spinal Cord
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Animals were anesthetized and sacrificed on day 21 after implantation of cancer cells. The sciatic nerve eroded by tumor mass and spinal cord were removed and postfixed at 4 o C for 5 h with 4% paraformaldehyde/PBS (pH7.4). The postfixed sciatic nerve and spinal cord were transferred to 15% sucrose/PBS for 24 h and then 30% sucrose/PBS for 24 h. The samples were frozen at -80 o C, serially cut with a cryostat (15 μm) and mounted on silane-coated slides.
After washing with ice cold PBS, sciatic nerve and spinal cord were blocked in solution containing 10 % normal goat serum and 0.1% Triton X-100 for 2 h at 4 o C. The sections were then incubated at 4 o C with primary antibodies against CD11b (1:1000; Cat# MCA711G, AbD Serotec, Oxford, UK), Iba1 (1:1,000; Cat# 01919741, FUJIFILM Wako Pure Chemical Tokyo, Japan), CD68 (1:500, Cat# 137001, BioLegend, San Diego, CA), or myelin-basic protein (1:500, Cat# 836504, BioLegend) for 48 h. After washing, the sections were incubated with a fluorescent-conjugated secondary antibody (Alexa 488, Alexa 546, Alexa 555 or Alexa 647, 1:1,000; Abcam, Cambridge, UK) at 4 o C for 2 h. The slides were covered with one drop of Vectashield (Vector Laboratories, Burlingame, CA), and then cover-slipped. Fluorescent images were obtained with confocal fluorescence microscopy.
After washing with ice cold PBS, sciatic nerve and spinal cord were blocked in solution containing 10 % normal goat serum and 0.1% Triton X-100 for 2 h at 4 o C. The sections were then incubated at 4 o C with primary antibodies against CD11b (1:1000; Cat# MCA711G, AbD Serotec, Oxford, UK), Iba1 (1:1,000; Cat# 01919741, FUJIFILM Wako Pure Chemical Tokyo, Japan), CD68 (1:500, Cat# 137001, BioLegend, San Diego, CA), or myelin-basic protein (1:500, Cat# 836504, BioLegend) for 48 h. After washing, the sections were incubated with a fluorescent-conjugated secondary antibody (Alexa 488, Alexa 546, Alexa 555 or Alexa 647, 1:1,000; Abcam, Cambridge, UK) at 4 o C for 2 h. The slides were covered with one drop of Vectashield (Vector Laboratories, Burlingame, CA), and then cover-slipped. Fluorescent images were obtained with confocal fluorescence microscopy.
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