Lungs from mice were formalin fixed and embedded in paraffin, and three 5-μm step sections were obtained for each lung. Sections were deparaffinized by standard treatment with xylene and ethanol. The following procedure was performed using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore, USA) according to the manufacturer’s instructions. Briefly, the tissue was pretreated with proteinase K (20 μg/ml), endogenous peroxidase was quenched by 3 % hydrogen peroxide for 5 min, and slides were incubated with TdT enzyme for 1 h at 37 °C. After washing, slides were incubated with anti-digoxigenin conjugate for 30 min at room temperature, washed and developed with peroxidase substrate. Slides were counterstained with methyl green and after dehydration mounted under glass coverslips using Permount media (Fisher Scientific, USA). Three photographs were taken from each slide and analyzed using ImageJ software.
Permount media
Manufactured by Thermo Fisher Scientific
Sourced in United States
Permount media is a mounting medium used in histology and microscopy for permanently mounting and preserving specimen slides. It is a xylene-based, synthetic resin that dries to a clear, hard finish, allowing for long-term storage and analysis of mounted samples.
Automatically generated - may contain errors
Lab products found in correlation
Aldehyde fuchsine solution, by Merck Group (2 mentions)
Alcian blue 8gx, by Merck Group (2 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Super coolscan 8000, by Nikon (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Light microscopy, by Leica (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
6 protocols using permount media
1
Apoptosis Detection in Mouse Lungs
2
Aldehyde Fuchsine Staining of Borrelia Aggregates
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The staining was performed as described previously with minor modifications [30 (link)]. Briefly, Borrelia aggregates on the deparaffinized and hydrated BL biopsy sections were stained with aldehyde fuchsine solution (Sigma-Aldrich, 0.5% fuchsine dye, 6% acetaldehyde in 70% ethanol with 1% concentrated hydrochloric acid) for 20 min. After immersing the slides in 70% ethanol for 1 min and having it rinsed with double-distilled water for another minute, the aggregates were sequentially stained with 1% Alcian blue 8GX (Sigma-Aldrich, dissolved in 3% acetic acid, pH 2.5) for 30 min. The slides were rinsed with double distilled water for 3 min and dehydrated using chilled graded ethanol washes (50%, 70%, and 95%, 3 min each). The slides were then immersed in chilled xylene for 2 min and mounted with Permount media (Fisher Scientific). Images were analyzed using different microscopy methods (see Results section).
3
Quantifying Spared White Matter Post-SCI
4
Leishmania Infection and Phagocyte Interaction
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One coverslip (Fisher) was added/well in 24 wells plate (BD Falcon). B10R (0.1 × 106 cells/1 ml of DMEM 10% FBS/well in 24 wells plate) and J774A.1 (0.2 or 0.1 × 106 cells/1 ml of RPMI 5% FBS/well in 12 wells plate) were plated on the day before the experiment. The next day medium was replaced by 300 μl of fresh medium containing Leishmania major (NIH S (MHOM/SN/74/Seidman) clone A2) or L. mexicana(MNYC/BZ/62/M379) in 1:20 (B10R) or 1:10 (J774A.1) ratios, assuming that cells had divided once since plating (i.e. considering double amount of cells compared to the starting numbers). After 6 h of infection, cells were washed with PBS and coverslips were put in presence of OlPC-liposome concentrations. Cells were incubated for an additional 18 hrs. After 24 h of culture, cells were washed twice with PBS and coverslips were collected and stained with Diff Quick (Behring #84132-1A). Subsequently dried coverslip were mounted on slides with Permount media (Fisher) and % of infected cells were determined using 3–5 count of 100 cells by light microscopy (Leica).
5
Biofilm Staining and Imaging Protocol
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The wild-type 297, RpoN, RpoS, and LuxS mutant strains of B. burgdorferi were incubated as described above for 7 days. These aggregates were fixed on the slides with chilled (-20 °C) 1:1 mixture of acetone–methanol for 5 min. Aldehyde fuchsine solution (Sigma-Aldrich, 0.5% fuchsine dye, 6% acetaldehyde in 70% ethanol with 1% concentrated hydrochloric acid) was used to stain the biofilm for 20 min at room temperature (RT). The slides were dipped in 70% ethanol for 1 min and then rinsed with double distilled water for 1 min followed by staining with 1% Alcian blue 8GX (Sigma-Aldrich, dissolved in 3% acetic acid, pH 2.5) for 30 min at RT. Then, after rinsing the slides with double distilled water for 3 min, they were dehydrated with graded ethanol (50%, 70%, and 95%, 3 min each), then dipped in chilled xylene for 2 min, and were mounted with Permount media (Fisher Scientific).
6
Silver Nitrate Staining Protocol
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One percent (1%) aqueous silver nitrate solution was made by adding 1 g of silver nitrate (cat 56506-256; Sigma) to 100 ml of distilled water. Five percent sodium thiosulfate solution was made by adding 5 g of sodium thiosulfate to 100 ml of distilled water. 0.1% nuclear fast red solution was made by adding 0.1 g nuclear fast red (cat N0305; TCI) and 5 g aluminum sulfate (cat A1114; Spectrum) to 100 ml distilled water and then boiled, cooled and filtered. A grain of thymol was added as a preservative. Sections were then warmed at 37℃ for 15 min, rinsed in PBS 2 × 5 min, and then rinsed in distilled water for 1 min. Sections were then incubated in 1% silver nitrate solution in a glass staining dish under ultraviolet light (Spectronics Spectrolinker XL-1000 UV Crosslinker) for 25 min. Slides were rinsed in distilled water for 1 min, then placed in 5% sodium thiosulfate, then rinsed in distilled water for 1 min. Slides were then counterstained in 0.1% nuclear fast red solution for 5 min and rinsed in distilled water. Slides were dehydrated 3 min in 95% EtOH and 2 × 3 min in 100% EtOH. Slides were then cleared in xylenes and coverslipped using Permount media (cat SP15-100; Fisher).
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