Tissue bisects were snap‐frozen in Tissue‐Tek optimum cutting temperature (OCT) compound (Sakura Finetek USA Inc., Torrance, CA) for cryo‐sectioning into 5 μm sections before staining for LDH activity in viable cells via precipitation of an insoluble purple‐blue formazan salt.34 After counterstaining with aqueous eosin, LDH‐positive cells appear dark blue. Histological slide sections were examined under a Nikon Ti‐S inverted microscope and scanned at ×4 using a slide scanner (PathScan Enabler 5, Meyer Instruments, Houston, TX). Slide scans were processed in FIJI35 using the ‘Freehand Line’ and ‘Freehand Selection’ tools to measure epidermal length and biopsy area, respectively. The red channel was adjusted using the ‘Colour Balance’ tool to increase contrast of the LDH stain before measuring, and the viable length and area were normalised to the total epidermal length and histological section area, respectively, of each biopsy.
Pathscan enabler 5
Manufactured by Meyer Instruments
The PathScan Enabler 5 is a benchtop instrument designed for the analysis of protein samples. It utilizes fluorescence-based detection technology to measure the activity of various signaling pathways and cellular processes.
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Lab products found in correlation
5 protocols using pathscan enabler 5
1
Quantifying Tissue LDH Activity
2
Quantification of Glandular Inflammation
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Quantitation of lacrimal and salivary gland inflammation was performed as previously described [53 (link)]. Briefly, exorbital lacrimal glands and submandibular salivary glands were fixed in formalin, processed, embedded in paraffin, and 5 µm sections were stained with hematoxylin and eosin (H&E). Inflammation was quantified in a blinded manner by standard light microscopy at 10× objective using standard focus-scoring with a focus defined as an aggregate of at least 50 mononuclear cells and the focus score defined as the number of foci per 4 mm2 of tissue. Tissue areas were calculated by ImageJ software [54 (link)] using low magnification digital images obtained by scanning H&E-stained sections with the PathScan Enabler IV (Meyer Instruments, Houston, TX, USA). The WT lacrimal and salivary glands in Figure 1 were previously published in comparison to another KO NOD strain [21 (link)] from the same colony as the Tlr7 KO samples to which they were compared in this study. Representative H&E-stained sections in the figure were obtained by whole-slide scans using the PathScan Enabler 5 (Meyer Instruments).
3
Porcine Skin Burn Histology Analysis
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Three uninjured normal porcine skin samples and 13 porcine skin samples previously identified as DPT burns were included in this analysis. Five labs contributed an early sample (0 hours after injury: n = 3 and 1 to 2 hours after injury: n = 4), and four labs contributed a late sample (24 hours after injury: n = 4, and 48 hours after injury: n = 2) and three labs contributed a normal, nonburned sample (n = 3). The entire slides of H&E stained tissue sections were imaged with the Aperio Digital Pathology Slide Scanner System (Leica Biosystems, Buffalo Grove, IL). The Aperio ImageScope pathology slide viewing software was used to visualize the entire high-resolution tissue section image file with magnification ranges of 2× to 40×. The EVG-stained slides were imaged with a 4× slide scanner to view the entire tissue sample using PathScan Enabler 5 (Meyer Instruments, Houston, TX). Image files were reviewed independently by eight authors in a blinded fashion, using the rubric usage guide (Supplementary Appendix B ) and revised scoring rubric (Supplementary Appendix C ).
4
Quantifying Cerebral Hemisphere Lesion
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To measure lesion volume, the serial brain sections were stained with hematoxylin and eosin Y (H&E), and digital images of the sections were acquired using a PathScan Enabler 5 (Meyer Instruments, Houston, TX). The lesion volume was calculated using ImageJ software (NIH, Bethesda, MD) and was expressed as the percentage of total ipsilateral and contralateral cerebral hemisphere volume.
5
Quantifying Inflammation in Lacrimal Glands
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Exorbital lacrimal glands were fixed in buffered formalin, processed, embedded in paraffin, sectioned, and stained with H&E. Inflammation was quantified by standard focus scoring [57 (link)]. Briefly, foci of inflammation (defined as aggregates with a minimum of 50 mononuclear cells) were counted in a blinded manner by standard light microscopy (10× objective). Sections were scanned using a PathScan Enabler IV (Meyer Instruments, Houston, TX, USA) to obtain a whole-section, low-resolution, digital images from which tissue section areas were measured using ImageJ software [62 (link)]. Inflammation was reported as focus score, which is equal to the number of inflammatory foci per 4 mm2 tissue area. Representative whole-section images in the figures were obtained with a PathScan Enabler 5 (Meyer Instruments).
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