Formalin fixed and paraffin embedded (FFPE) tissue specimens were sliced in 4 μm sections using a Leica RM2235 manual microtome. Dried sections were de-waxed by immersion in xylene and rehydrated with subsequent immersion in 100% ethanol, 70 % ethanol and distilled water. Antigen retrieval was performed by immersion in PBS 0.01 M citric acid pH 6 and heated at 800 W for 15 min. Slides were rinsed in PBS and endogenous peroxidase activity was blocked for 30 min using Dako RealTM Peroxidase Blocking Solution. Following that, slides were rinsed twice with PBS and incubated with 10% pig serum (Bio-Rad) for 30 min and overnight with KRT80 antibody (Sigma-Aldrich, 1:200). Following day, slides were rinsed in PBS and incubated 30 min with secondary antibody (biotinylated Goat Anti-Rabbit IgG 1:200, Vector Laboratories) and 30 min with an avidin/biotin peroxidase-based system (VECTASTAIN Elite ABC Kit, Vector Laboratories). Color reaction was developed for 1 min using DAB (Diaminobenzidine, Vector ImmPACT DAB Peroxidase Substrate). Color development was stopped by immersion during 5 min in running tap water and following that, nuclei was stained with haematoxylin. Slides were dehydrated in 100% ethanol, cleared in xylene and mounted in DPX (SIGMA).
Rm2235 manual microtome
Manufactured by Leica
The RM2235 is a manual microtome, a laboratory instrument used for cutting thin sections of various materials for microscopic examination. It features a vertical feed mechanism and a specimen orientation system to allow precise positioning of the sample.
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Lab products found in correlation
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Pig serum, by Bio-Rad (1 mentions)
2 protocols using rm2235 manual microtome
1
Immunohistochemical detection of KRT80
2
Tissue Sectioning and Slide Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The microtome used for sectioning was a Leica RM 2235 manual microtome. The refrigerated tissue blocks of the vehicle-, fluoxetine-, desferrioxamine- and iron-treated groups were mounted on the microtome, sectioned at 50 and placed on water. The floating sections were picked with a brush and mounted on the gelatine coated slides. The sections were blotted with tissue paper and direct, downward moderate pressure was applied with the heel of the palm (Gibb and Kolb, 1998 (link)) so that the sections were firmly glued to the gelatine slides. The slides with sections were transferred to racks and kept for drying in the dark for 3 days.
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