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348 protocols using eclipse c1

1

Histological Analysis of Organ Tissue

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On day 10, the mice in each group were sacrificed.
For H&E staining, the major organ of mice was collected, fixed
in 4% paraformaldehyde overnight, and embedded in paraffin according
to standard histological protocols. Next, the tissue was cut into
5 μm-thick cross-sectional slices. The paraffin sections in
each group were dewaxed, stained with hematoxylin for 3–5 min,
and differentiated with ethanolic hydrochloric acid. The paraffin
sections were washed and stained with eosin for 5 min. Images were
photographed under a microscope (Eclipse C1, Nikon, Japan).
For TUNEL staining, the harvested tumor tissues were first fixed
with 4% paraformaldehyde. Then, they were embedded in an optimal cutting
temperature compound followed by cutting into 5 μm-thick sections.
The sections were treated using Triton X-100 at room temperature for
10 min and then blocked with 5% bovine serum albumin. The apoptotic
cells were measured with a TUNEL apoptosis assay kit in accordance
with the manufacturer’s instruction. DAPI was selected for
nuclear staining. The image was photographed under a microscope (Eclipse
C1, Nikon, Japan).
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2

Apoptosis and Nissl Staining in Rat Cortex

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Based on the manufacturer’s instructions, we employed the TUNEL assay kit (G1502, Servicebio, China) for labeling paraffin-embedded sections in order to identify apoptotic cells in the cortical tissue. The TUNEL kit was marked with CY3 fluorescein, resulting in the visualization of red-stained positive apoptotic cell nuclei, while DAPI was employed to stain regular cell nuclei, which appeared in blue. The apoptotic index was determined as the ratio of TUNEL-positive nuclei to the total number of nuclei. The quantification of TUNEL + cells was carried out using a microscope (Nikon Eclipse C1; Nikon, Japan) and analyzed with NIH ImageJ Software (Bethesda, MD, USA) by a technician who was unaware of the experimental design. For each Rat sample, a minimum of three slides were stained and evaluated for cell counting. For Nissl staining, the sections were immersed in Nissl staining solution (G1036, Servicebio) for 4 minutes, followed by rinsing with double-distilled water. Subsequently, a slight differentiation was achieved using 0.1% glacial acetic acid. After thorough water washing, the sections were air-dried in an oven, dehydrated with xylene until transparent (approximately 10 minutes), and finally mounted with neutral gum. Photomicrographs were acquired using a light microscope (Nikon Eclipse C1; Nikon, Japan).
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3

Measuring Calcium Dynamics in Olfactory Astrocytes

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Slices were incubated with the membrane-permeable form of the calcium indicator Fluo-8 (Fluo-8-AM; 2 μM in ACSF) made from a 2 mM stock solution (dissolved in DMSO and 20% pluronic) for 30 min. Brain slices were then placed in the recording chamber and fixed with a U-shaped platinum wire with nylon strings. Brain slices were continuously perfused at a rate of 2 ml/min with ACSF that was gassed with carbogen (95 O2/5% CO2). Bath perfusion with ACSF was accomplished using a peristaltic pump (Vario, Ismatec, Germany). Drugs were applied via the perfusion system. If not stated otherwise, ACSF contained 1 μM TTX to suppress neuronal activity. Changes in intracellular calcium levels in olfactory bulb astrocytes were recorded by confocal microscopy (C1 Eclipse, Nikon, Düsseldorf, Germany). An excitation laser wavelength of 488 nm and a frame rate of 0.66 fps were used, and the fluorescence was collected through a 500–530 nm bandpass filter.
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4

Astrocytic Calcium Dynamics in Olfactory Bulb

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Slices were incubated with the membrane-permeable form of the calcium indicator Fluo-8 (Fluo-8-AM; 2 µM in ACSF) made from a 2 mM stock solution (dissolved in DMSO and 20% pluronic acid) for 30 min. Brain slices were then placed in the recording chamber and fixed with a U-shaped platinum wire with nylon strings. Brain slices were continuously superfused at a rate of 2 ml/min with ACSF that was gassed with carbogen (95 O2/5% CO2). Bath perfusion with ACSF was accomplished using a peristaltic pump (Vario, Ismatec, Germany). Drugs were applied via the perfusion system. If not stated otherwise, ACSF contained 1 µM TTX to suppress neuronal activity. Changes in intracellular calcium levels in olfactory bulb astrocytes were recorded by confocal microscopy (C1 Eclipse, Nikon, Düsseldorf, Germany). An excitation laser wavelength of 488 nm and a frame rate of 0.75 fps were used and the fluorescence was collected through a 500–530 nm bandpass filter.
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5

Immunohistochemical Analysis of Kidney Tissue

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Kidneys were fixed in 4% PLP (4% paraformaldehyde, 75 mM l-lysine, 10 mM sodium periodate) for 2 h at 4 °C, and then placed in 30% sucrose overnight. Tissues were snap frozen in optimal cutting temperature compound (OCT, Sakura FineTek, Torrance, CA) and cryosections of 7 μM were mounted on microscope slides. Sections were incubated overnight with primary antibodies as indicated: anti-F4/80 (hybridoma supernatant) and anti-GR1 + (eBioscience), or anti-Kidney-specific cadherin (Ksp-cadherin) (Morizane et al., 2013 (link)). Slides were then incubated with Cy3 or FITC labeled secondary antibodies (Jackson ImmunoResearch). Sections were mounted in Vectashield containing DAPI to stain the nuclei (VectorLabs, Burlingame, CA). Neutrophils were expressed as the mean number of GR1 + cells per 400 × magnification field and macrophages as the mean per unit area of F4/80 + cells in 400 × magnification fields using ImageJ. Ksp-cadherin positive area was also expressed as the mean of KSP stain positive area in 200 × magnification fields. Ten randomly selected images per mouse were quantified using ImageJ software (http://rsbweb.nih.gov/ij/) (Schrimpf et al., 2012; Grgic et al., 2012 ). All images were obtained by standard or confocal microscopy (Eclipse 90i, C1 Eclipse, respectively; both from Nikon).
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6

Immunofluorescence Staining Procedure

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Immunofluorescence staining on the cells was performed as previously described [33 (link),34 (link),35 (link)]. Briefly, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Samples were permeabilized using 1% Triton X-100 for 15 min for nuclear and cytoplasmic staining at room temperature or left unpermeabilized for the membrane protein. The cells were washed twice with PBS and blocked with two percent normal sheep serum containing 5% BSA for 1 h at room temperature. The primary antibodies (1:200 dilutions) were incubated overnight at 4 °C in five times diluted blocking buffer containing 0.1% Tween 20. After three washes of PBST (0.1% Tween 20), fluorescence-labeled species-specific secondary antibodies (Thermo Fisher Scientific) were added and incubated for 1 h at room temperature. The slides were washed thrice with PBST and mounted with a medium containing DAPI (Vector laboratories). Three to five images were captured using a 60× objective on a Nikon Confocal Imaging system (Nikon C1 Eclipse, Nikon, Melville, NY, USA).
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7

Immunofluorescence Staining of Kidney Cells

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Immunofluorescence protocols and antibodies are detailed below. Frozen sections were used for immunofluorescence staining. Primary cultured tubular epithelial cells on Matrigel-coated coverslips and kidney sections were permeabilized with 0.2% Triton X-100 and fixed with 4% (vol/vol) paraformaldehyde for 5 min. Samples were blocked with 5% (vol/vol) normal goat serum in PBS and incubated with primary antibodies including FITC-conjugated anti-lotus LTL (FL-1321; Vector Labs; 1:1,000), goat anti-α-smooth muscle actin (SMA) (1:1,000, Abcam, ab21027), goat anti-kidney injury molecule-1 (Kim-1) (AF1817; R&D Systems; 1:500), rabbit anti-Pax2 (ab92547; Abcam; 1:500), and rabbit anti-vimentin (#5741; CST; 1:500). Secondary antibodies were either FITC- or Cy3-conjugated (Jackson ImmunoResearch) and incubated for 1 h. Nuclear counterstaining was performed using DAPI (Invitrogen). Images were obtained by confocal (Nikon C1 Eclipse; Nikon) or standard (Nikon Eclipse 90i; Nikon) microscopy.
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8

Immunohistochemical Analysis of CD68

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Patient tissues or wound-edge skin tissues from mice were fixed in 4% paraformaldehyde solution and dehydrated in an ethanol gradient. After clearing with xylene, tissue samples were embedded in paraffin and cut into histological sections (RM2016, Shanghai Leica Instrument Co., Ltd. Shanghai, China). The slides were then incubated with a primary antibody (CD68; Affinity, USA), followed by incubation with a goat anti-rabbit secondary antibody. Images were obtained using a confocal microscope (Nikon C1 Eclipse; Nikon).
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9

Sorafenib-Induced Apoptosis Pathway Analysis

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Cells were plated on a multi-chamber slide (MilliporeSigma) and treated with 0.1 µM sorafenib for 72 h. Immunofluorescence staining was performed as previously described. In brief, cells were fixed with 4% PFA for 15 min and permeabilized using 1% Triton X-100 for 15 min. Two percent normal sheep serum containing 5% BSA was used for blocking for 1 h. Primary antibodies (anti-Caspase 3, dilution of 1:100, Cell Signaling Technology; anti-FasL, dilution of 1:50, Abcam; anti-Bax, dilution of 1:50 Santacruz Biotechnology; anti-p53, dilution of 1:100, MilliporeSigma) were added and incubated overnight at 4 °C in 5x diluted blocking buffer containing 0.1% Tween 20. After three washes with PBST (0.1% Tween 20), Alexa Fluor 488 or Alexa Fluor 595 labeled secondary antibodies (Thermo Fisher Scientific) were added and incubated for 1 h at room temperature. Slides were washed and mounted using a medium containing DAPI. Images were captured on a Nikon Confocal Imaging system (Nikon C1 Eclipse, Nikon, Tokyo, Japan).
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10

Sorafenib-Induced Apoptosis Assay

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Cells were plated on a multi-chamber slide (MilliporeSigma) and treated with 0.1 µM sorafenib for 72 h. Cells were incubated with Alexa488 labeled Annexin V reagent (Thermo Fisher Scientific) for 1 h. Cells were then fixed with 4% PFA, washed with PBST three times, and mounted with DAPI-containing medium. Images were captured on a Nikon Confocal Imaging system (Nikon C1 Eclipse, Nikon).
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