Paraffin-embedded liver sections, 40 μm per section, from male 16-wk-old R6/1 mice containing the Cre and floxed alleles of IKKβ (HD), treated with tamoxifin (n = 5) or oil vehicle control (n = 4), were used. One section per mouse was analyzed. Fluorescent aggregate immunolabeling was done with anti-HTT MW8 (AB528297; Developmental Studies Hybridoma Bank) (recommendation from Jeffrey Carroll for liver HTT, Western Washington University, Bellingham, WA) and TO-PRO-3 Iodide stain (T3605; Thermo Fisher Scientific) was used to label nuclei. Images were captured using Leica confocal microscope (DM2500) and Leica camera TCS SPE (Leica Microsystems Inc.). All images were taken at 20×, two areas per section, with consistent settings for laser intensity, exposure time, and gain through all images. In each area, the number of aggregates was counted by eye without normalization, as aggregate numbers were very low and visible per field.
Camera tcs spe
Manufactured by Leica
The Leica camera TCS SPE is a high-performance confocal microscope designed for advanced fluorescence imaging. The instrument features a modular and flexible design, allowing for customization to meet the specific needs of various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti htt mw8, by Developmental Studies Hybridoma Bank (1 mentions)
Shandon polysine slides, by Thermo Fisher Scientific (1 mentions)
Dpx mounting medium, by Electron Microscopy Sciences (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Mem48, by Merck Group (1 mentions)
4 protocols using camera tcs spe
1
Quantifying Huntingtin Aggregates in Liver
2
Fluoro-Jade B Staining of Striatal Sections
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Analysis was done using four male postfixed half-brains for each treatment group, with five 40-μm representative sections prepared from each. Stack images (20×) were obtained using confocal microscopy at comparable sections for each animal. Images were captured using Leica confocal microscope (DM2500) and Leica camera TCS SPE (Leica Microsystems Inc.). Striatal sections were mounted on Shandon Polysine slides (Thermo Scientific), allowed to dry completely, and immersed in a series of washes: (i) 3 min in 80% ethanol, (ii) 2 min in 70% ethanol, (iii) 2 min in a 1:200 dilution of acetic acid to MilliQ water, (iv) 10 min gently rocking in 0.06% KMnO4 solution, (v) 2 min in a 1:200 dilution of acetic acid to MilliQ water, (vi) 15 min gently rocking in 0.0004% Fluoro-Jade B (AG310; Millipore Sigma) solution, and (vii) three sequential 1-min rinses in a 1:200 dilution of acetic acid to MilliQ water. Slides were allowed to dry, immersed in three sequential 1-min washes with 100% xylene, allowed to dry again, and coverslipped with DPX mounting medium (Electron Microscopy Sciences).
3
Quantifying Microglia in Mouse Brains
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For immunohistochemical assessments, 40-μm sections of postfixed half-brains from four 16-wk-old male mice per treatment group were processed for immunohistochemistry and imaged via confocal microscopy. The following primary antibody was used: anti-Iba1 (27030; Wako Pure Chemicals Industries). Alexa fluorescent-conjugated secondary antibody was used (A21428; Thermo Fisher Scientific). Coronally sectioned stained tissue was processed and mounted on slides with Fluoromount-G (SouthernBiotech). Images were captured using Leica confocal microscope (DM2500) and Leica camera TCS SPE (Leica Microsystems Inc.). Five comparable representative images per treatment group were taken at 10×, 20×, and 63× z stack, with consistent settings for laser intensity, exposure time and gain through all images. Automatic analyses and cell quantitation from acquired images was processed using Imaris x64 quantitation software (Bitplane Scientific Software).
4
Quantifying Huntington's Disease Protein Aggregates
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Half-brain coronal sections, 40 μm each, four sections per 16-wk-old male mouse, were used for quantitation. Sections were picked from bregma 1.34, every 12 sections, to bregma −0.1, from male R6/1 mice containing the Cre and floxed alleles of IKKβ (HD) and treated with tamoxifin or oil vehicle control, four mice per treatment group. Fluorescent immunolabeling was done with anti-HTT clone mEM48 (MAB5374; Millipore) to label transgenic human mutant HTT exon 1 protein aggregates and TO-PRO-3 Iodide stain (T3605; Thermo Fisher Scientific) to label nuclei. Images were captured using Leica confocal microscope (DM2500) and Leica camera TCS SPE (Leica Microsystems Inc.). Images were taken at 40×, with z-volume set at 25 to 30 μm at 1 μm per step. Three striatal areas, upper, middle and lower, were imaged with consistent settings for laser intensity, exposure time, and gain through all images. Imaris x64 quantitation software (Bitplane Scientific Software) was used to evaluate the number of aggregates in each area normalized to the number of nuclei, calculated as the percent of cells expressing aggregates.
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