It was performed as described before.60 (link),63 (link) Briefly, mice were anesthetized and perfused with PBS (pH 7.4) and then with 4% (w/v) paraformaldehyde solution in PBS followed by dissection of the brain from each mouse for IHC.64 (link),65 (link) Samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 h and then 30% sucrose overnight at 4°C. Brain was then embedded in O.C.T (Tissue Tech) at −80°C, and processed for conventional cryosectioning. Frozen sections (30 μm) were treated with cold ethanol (−20°C) followed by two rinses in PBS, blocking with 3% BSA in PBST and double labeling with two antibodies (Table S1 ). After three washes with PBST, sections were further incubated with Cy2 or Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). The samples were mounted and observed under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera. During Thio-S staining, after washing and incubation with secondary antibodies for 1 h, free-floating sections were stained with 0.002% Thio-S (Sigma) made up in TBS for 8 min. Sections were washed twice in 50% EtOH for 1 min and twice in TBS for 5 min before drying and mounting in Fluoromount (Sigma).
Thio s
Manufactured by Merck Group
Sourced in United States
Thio-S is a laboratory equipment product manufactured by Merck Group. It is a device used for the detection and analysis of sulfur-containing compounds. The core function of Thio-S is to provide accurate and reliable measurements of sulfur content in various samples, enabling researchers and analysts to better understand the composition and properties of their materials.
Automatically generated - may contain errors
Lab products found in correlation
Bx41 fluorescent microscope, by Hamamatsu Photonics (2 mentions)
Orca 03g camera, by Hamamatsu Photonics (2 mentions)
Cy2 or cy5 conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Fluoromount, by Merck Group (2 mentions)
Superfrost plus slides, by Avantor (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Gold seal rite on ultra frost, by Thermo Fisher Scientific (2 mentions)
Capsure lcm macro caps, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm900 confocal laser scanning microscopy, by Zeiss (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
Anti rabbit txrd, by Southern Biotech (1 mentions)
Anti neun, by Merck Group (1 mentions)
Dp74 camera, by Olympus (1 mentions)
Vibratome, by Leica (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
Anti gfap, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
21 protocols using thio s
1
Immunofluorescence and Thioflavin-S Staining in Mouse Brain
2
Immunofluorescence and Thioflavin-S Staining in Mouse Brain
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It was performed as described before.60 (link),63 (link) Briefly, mice were anesthetized and perfused with PBS (pH 7.4) and then with 4% (w/v) paraformaldehyde solution in PBS followed by dissection of the brain from each mouse for IHC.64 (link),65 (link) Samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 h and then 30% sucrose overnight at 4°C. Brain was then embedded in O.C.T (Tissue Tech) at −80°C, and processed for conventional cryosectioning. Frozen sections (30 μm) were treated with cold ethanol (−20°C) followed by two rinses in PBS, blocking with 3% BSA in PBST and double labeling with two antibodies (Table S1 ). After three washes with PBST, sections were further incubated with Cy2 or Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). The samples were mounted and observed under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera. During Thio-S staining, after washing and incubation with secondary antibodies for 1 h, free-floating sections were stained with 0.002% Thio-S (Sigma) made up in TBS for 8 min. Sections were washed twice in 50% EtOH for 1 min and twice in TBS for 5 min before drying and mounting in Fluoromount (Sigma).
3
Multimodal Imaging of Neuroinflammation
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Fluorescent immunolabeling was performed using a standard indirect technique as described previously [22 (link)]. Brain sections were stained with primary antibodies against: ionized calcium binding adaptor molecule 1 (IBA1; 1:1000; 019-19741, Wako and ab5076, Abcam), CD68 (1:200; BioRad) glial fibrillary protein (GFAP; 1:1000; Abcam), NeuN (1:1000; Millipore), OLIG2 (1:200; Abcam), Aβ1–16 (6E10; 1:1000; Biolegend), and anti-lysosomal associated membrane protein 1 (LAMP1; 1:200; Santa Cruz Biotechnologies). For Amylo-Glo staining (TR-300-AG; Biosensis), tissue sections were washed in 70% ethanol 1 × 5 min, followed by a 1 × 2 min wash in distilled water. Sections were then placed in a 1% Amylo-Glo solution for 1 × 10 min then washed with 0.9% saline for 1 × 5 min and distilled water for 1 × 15 s before continuing fluorescent immunolabelling. For Thioflavin-S (Thio-S) staining, tissue sections were placed for 1 × 10 min incubation in 0.5% Thio-S (1892; Sigma-Aldrich) diluted in 50% ethanol. Sections were then washed 2 × 5 min each in 50% ethanol and one 10-min wash in 1xPBS before continuing with fluorescent immunolabelling.
High resolution fluorescent images were obtained using a Leica TCS SPE-II confocal microscope and LAS-X software. For confocal imaging, one field of view (FOV) per brain region was captured per mouse unless otherwise indicated.
High resolution fluorescent images were obtained using a Leica TCS SPE-II confocal microscope and LAS-X software. For confocal imaging, one field of view (FOV) per brain region was captured per mouse unless otherwise indicated.
4
Amyloid-β Plaque Quantification in Hippocampus
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Two slides were randomly selected from the hippocampus-bearing tissue collections of each mouse (n = 5 each), deparaffinized in xylene, hydrated by a decreasing ethanol gradient series, and washed twice in distilled water. The sections were incubated with 1% thioflavin S (Thio-S; Sigma-Aldrich), a specific staining tool for the amyloid β plaques, and dissolved in 50% ethanol at 22 °C for 10 min. Coverslips were mounted on slides and observed under an LSM900 confocal laser scanning microscopy (Zeiss). Under a fluorescence illumination setting (excitation/emission wavelength: 391/428 nm), both hippocampi were photographed and the total fluorescence areas in a high-power field (HPF, X200) were averaged.
5
Thioflavin S Staining of Tissue Sections
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The prepared slices were soaked in 1% ThioS (Sigma) dissolved in 50% ethanol in the dark for 8 min. After that, the sections were washed twice with 50% ethanol for 10 s each time, and finally with distilled water for 5 min. The results were observed using confocal microscopy and analyzed with image J software.
6
NeuN and Thioflavin S Staining Protocol
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ThioS stain was done as previously described 62 (link). Briefly, slices were washed twice for 10 min in PBS. Next, slices were put in a blocking solution (TBS, 0.3% TritonX-100, 3% goat serum) for 2 h. Then slices were put in anti-NeuN (Millipore, ABN78) 1:1000 in TBS with 0.3% Triton X-100 (TBS-T) overnight. The next day, slices were washed three times in TBS-T, then put in anti-rabbit-TXRD (Southern Biotech, 4010-07) 1:300 in TBS-T overnight. The next day, 3 TBS-T washes were performed, then the slices were stained in 1% ThioS (Sigma, T1892) for 9 min. To de-stain, slices were washed twice with water, once with 70% ethanol, then twice with water. Slices were finally washed again in TBS then mounted onto Superfrost Plus slides (VWR, 48311-703). AF1+DAPI (EMS, 17970-125) mounting media was added, then the coverslip. Slides were imaged using a Zeiss Axio Imager.M2 the next day.
7
Quantifying Amyloid Plaques in Brain
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Fixed hemi-brains were fully sectioned in the horizontal plane at 40 μm using a vibratome (Leica Biosystems). Every eighth section was stained for Thio-S (#230456, Sigma-Aldrich) using standard methodology. Sections were mounted and allowed to dry overnight, after which they were washed three times in 50% ethanol for 5 min each, then washed in double-distilled H2O before being incubated for 10 min in 1% Thio-S dissolved in H2O. Stained slides were then rinsed in 70% ethanol before being dehydrated and coverslipped in aqueous anti-fade mounting medium (Vector Laboratories). Digital images were captured at 20× magnification using an Olympus BX50 microscope equipped with a DP74 camera and CellSens software (Olympus). The number of spherical thioflavin-positive deposits were counted using NIH ImageJ 1.50i (United States National Institutes of Health) with the cell counter plugin to mark stained plaque-like structures. Thioflavin-positive deposits were counted in entorhinal cortex (three fields/section), subiculum (two fields/section), and hippocampal subfields CA1 (three fields/section) and CA2/3 (three fields/section), across four sections per animal, for a total of ∼44 fields per brain.
8
GFAP Immunostaining and ThioS Staining
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Slices were washed twice for 10 min in PBS. Next, slices were put in a blocking solution (TBS, 0.3% Triton X-100, 3% goat serum) for 2 h. Then slices were put in anti-GFAP (Invitrogen, PA5-16291) 1:1000 in TBS-T for two days. Next, slices were washed three times in TBS-T, then put in anti-rabbit-594 (Abcam, ab96985) 1:500 in TBS-T overnight. The next day, 3 TBS washes were performed, then the slices were stained in 1% ThioS (Sigma, T1892) for 9 min. To de-stain, slices were washed twice with water, once with 70% ethanol, then twice with water. Slices were finally washed again in TBS then mounted onto Superfrost Plus slides (VWR, 48311-703). AF1+DAPI (EMS, 17970-125) mounting media was added, then the coverslip. Slides were imaged using a Zeiss Axio Imager.M2 the next day.
9
Thioflavin S Staining of Amyloid Plaques
10
Fluorescent Staining of Brain Sections
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