Immunohistochemical analyses were carried as previously described (17 (link)). Briefly, colonic tissue sections were deparaffinized, hydrated, followed by antigen retrieval, blocking and overnight incubation with anti-Ly-6B.2 antibody (BioRad Laboratories Inc.). Signals were visualized using the Vectastain ABC and DAB kits (Vector Laboratories) and haematoxylin counterstaining. Images were taken using the Lecia DM2000 LED light microscope (Chicago, IL, USA). Neutrophil infiltration scores were assessed blindly using the criteria described previously (18 (link)). Briefly, the scores for the depth of neutrophil infiltration (scored as 0–3) and staining intensity (0–4) were recorded individually. The summation of both scores resulted in the total quantified score ranging from 0 to a maximum of 7 per distal colonic section. Nine sections per animal at constant interval were used for microscopic examination and score assessment.
Dm2000 led light microscope
Manufactured by Leica camera
Sourced in United States
The Leica DM2000 LED is a light microscope that utilizes LED illumination. It is designed for professional laboratory use and offers high-quality optics for detailed observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti ly 6b 2 antibody, by Bio-Rad (3 mentions)
Vectastain abc and dab kits, by Vector Laboratories (2 mentions)
Methyl salicylate, by Merck Group (2 mentions)
C11440 camera, by Hamamatsu Photonics (1 mentions)
Dmc2900 camera, by Leica camera (1 mentions)
Eclipse ni light microscope, by Nikon (1 mentions)
D6b fluorescence microscope, by Leica camera (1 mentions)
Dfc9000 gt camera, by Leica camera (1 mentions)
Microfil, by Flow Tech (1 mentions)
9 protocols using dm2000 led light microscope
1
Quantifying Neutrophil Infiltration in Distal Colon
2
Quantitative Analysis of Fluo-CQ Uptake
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Giemsa-stained culture smears were imaged on a DM2000 LED light microscope (Leica) equipped with a DMC2900 camera (Leica). Fluorescence microscopy was performed with an Eclipse Ni light microscope (Nikon) fitted with a C11440 camera (Hamamatsu) or with a D6B fluorescence microscope (Leica) equipped with a DFC9000 GT camera (Leica). For detailed microscopy protocols including quantitative analysis of Fluo-CQ uptake, please refer to SI Appendix .
3
Quantifying Neutrophil Infiltration in Distal Colon
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Immunohistochemical analyses were carried as previously described (17 (link)). Briefly, colonic tissue sections were deparaffinized, hydrated, followed by antigen retrieval, blocking and overnight incubation with anti-Ly-6B.2 antibody (BioRad Laboratories Inc.). Signals were visualized using the Vectastain ABC and DAB kits (Vector Laboratories) and haematoxylin counterstaining. Images were taken using the Lecia DM2000 LED light microscope (Chicago, IL, USA). Neutrophil infiltration scores were assessed blindly using the criteria described previously (18 (link)). Briefly, the scores for the depth of neutrophil infiltration (scored as 0–3) and staining intensity (0–4) were recorded individually. The summation of both scores resulted in the total quantified score ranging from 0 to a maximum of 7 per distal colonic section. Nine sections per animal at constant interval were used for microscopic examination and score assessment.
4
Histological Evaluation of Colonic Inflammation and Adipose Tissue Analysis
5
Quantifying Neutrophil Infiltration in Distal Colon
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Paraffin embedded tissues were cut into 5 µm thick sections, deparaffinized, and hydrated, followed by antigen retrieval, goat serum blocking, and overnight incubation with anti-Ly-6B.2 antibody (Bio-Rad Laboratories Inc., Hercules, CA, USA). After incubation with the secondary antibody, signals were visualized using the Vectastain ABC and DAB peroxidase (HRP) substrate kits (Vector Laboratories Inc., Burlingame, CA, USA) and haematoxylin counterstaining. Images were taken using the Lecia DM2000 LED light microscope (Chicago, IL, USA). Neutrophil infiltration scores were assessed blindly by two researchers using the criteria described previously [20 (link)]. Briefly, the scores for depth of neutrophil infiltration (scored as 0–3) and staining intensity (scored as 0–4), which was the percent area positive as extent (0, none; 1, <25%; 2, 25–50%; 3, 50–75%; 4, >75%), were recorded individually. The summation of both scores resulted in a total quantified score ranging from 0 to a maximum of 7 per distal colonic section. Nine sections per animal at constant interval were used for microscopic examination and score assessment.
6
Histological Evaluation of Colonic Inflammation and Adipose Tissue Analysis
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Paraffin embedded tissues were sectioned at 5 μm thickness, deparaffinized and subjected to haematoxylin and eosin (H&E) staining. Histological examination and imaging were done under a Lecia DM2000 LED light microscope (Chicago, IL, USA). At least one image was obtained per section and 9 sections per animal at constant interval were used for microscopic examination.
The pathological score of the distal colon was evaluated and recorded blindly using previously published score criteria (15 (link)), which was the sum of the scores of crypt damage (none, basal 1/3, basal 2/3, only surface epithelium intact), severity of inflammation (none, slight, moderate, and severe), and depth of injury (none, mucosal, mucosal and submucosal, and transmural). Paraffin embedded adipose tissues were sectioned and processed as previously described (16 (link)). Image J 1.30v software (National Institute of Health, Bethesda, MD, USA) was used to measure the area and diameter of adipocytes by drawing a horizontal straight edge-to-edge line in the middle of the adipocyte. Nine sections per animal at constant interval were used and all the adipocytes per image were quantified.
The pathological score of the distal colon was evaluated and recorded blindly using previously published score criteria (15 (link)), which was the sum of the scores of crypt damage (none, basal 1/3, basal 2/3, only surface epithelium intact), severity of inflammation (none, slight, moderate, and severe), and depth of injury (none, mucosal, mucosal and submucosal, and transmural). Paraffin embedded adipose tissues were sectioned and processed as previously described (16 (link)). Image J 1.30v software (National Institute of Health, Bethesda, MD, USA) was used to measure the area and diameter of adipocytes by drawing a horizontal straight edge-to-edge line in the middle of the adipocyte. Nine sections per animal at constant interval were used and all the adipocytes per image were quantified.
7
Apoptosis Detection in Gut Tissue
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In situ apoptosis was determined using TACS TdT-DAB detection kit (Trevigen, Gaithersburg, MD, USA) per the manufacturer's instructions. Briefly, ex vivo gut sections were treated with Cytopore and blocked with 3% H2O2. DNA fragments were labelled with biotin and HRP-conjugated streptavidin, then incubated with DAB substrates. The counterstaining was performed with 1% Methyl Green. The images were taken using Leica DM2000 LED light microscope (Wetzlar, Germany).
8
Delayed Vasospasm in SAH Mice
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Previous studies from our lab show the presence of vascular spasm in the middle cerebral artery (MCA) of SAH mice early (at day 1) and, more importantly, in a delayed manner (6 days after the hemorrhage) corresponding to DCI in mice (5) (link). Since the peak neutrophil in ltration occurs in our model 3 days posthemorrhage and delayed vasospasm is correlated with DCI, we focused our vasospasm analysis on the delayed vasospasm occurring on the 6 th day after SAH.
The arterial tree of each mouse was labeled using the vascular dye Micro l (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Brie y, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Micro l dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to nonuniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest cross-sectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.
The arterial tree of each mouse was labeled using the vascular dye Micro l (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Brie y, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Micro l dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to nonuniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest cross-sectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.
9
Delayed Cerebral Vasospasm in SAH Mice
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Previous studies from our lab show the presence of vascular spasm in the middle cerebral artery (MCA) of SAH mice early (at day 1) and, more importantly, in a delayed manner (6 days after the hemorrhage) corresponding to DCI in mice (5) (link). Since the peak neutrophil infiltration occurs in our model 3 days post-hemorrhage and delayed vasospasm is correlated with DCI, we focused our vasospasm analysis on the delayed vasospasm occurring on the 6 th day after SAH.
The arterial tree of each mouse was labeled using the vascular dye Microfil (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Briefly, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Microfil dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to non-uniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest crosssectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.
The arterial tree of each mouse was labeled using the vascular dye Microfil (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Briefly, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Microfil dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to non-uniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest crosssectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.
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