Histological confirmation of cannula placements in the BNST for drug microinjection was performed after the behavioral experiments. Mice were transcardially perfused with ice-cold 0.1 M phosphate-buffered saline (PBS) for 15 min, followed by filtered ice-cold 4% paraformaldehyde (PFA) in 0.1 M PBS for 15 min. The brains were carefully removed and further post-fixed in filtered 4% PFA for at least 24 h, then 30% sucrose at 4 °C for at least 48 h. Afterward, 40-µm-thick sections were cut from the frozen brain block, using a cryostat at −15 °C (Leica CM1100; Leica Biosystems, Nußoch, Germany), and mounted on poly-L-lysine-coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan; Cat. # S7441). After washing with PBS for 5 min, the sections were incubated with Mayer's Hematoxylin Solution (Wako Pure Chemical Industries Ltd.; Cat. # 131–09665) at room temperature (25 ± 2 °C) for 5 min. The hematoxylin-stained sections were washed with 50 °C water for 5 min. Images were captured using an 8-LED USB Digital Microscope Endoscope Magnifier Camera (FB-CMXW02; Koolertron, Shenzhen, China) equipped with Micro Capture Pro software (Celestron, LLC, Torrance, CA, USA). Probe placements were verified by adapting the Paxinos mouse brain atlas [34] , mainly for the dorsal and ventral BNST (Figure 1B and C
Poly l lysine coated glass slides
Manufactured by Matsunami
Sourced in Japan
Poly-L-lysine-coated glass slides are a type of laboratory equipment used for various applications. They provide a positively charged surface that can facilitate the attachment and adhesion of cells, tissues, or other biological samples during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cellomics arrayscan vti hcs reader, by Thermo Fisher Scientific (2 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
Cm1100, by Leica (1 mentions)
Envision hrp detection system, by Agilent Technologies (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Liquid dab chromogen, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
A4018, by Merck Group (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
3 3 diaminobenzidine dab substrate kit, by Nichirei Biosciences (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Pentobarbital sodium, by Kyoritsu Seiyaku (1 mentions)
9 protocols using poly l lysine coated glass slides
1
Histological Verification of BNST Cannula Placements
2
Immunohistochemical Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibody sources used in this study are shown in Table 2 . Paraffin sections were cut (3 μm) and mounted on poly-l -lysine–coated glass slides (Matsunami, Tokyo, Japan). Sections were routinely dewaxed and dehydrated and then subjected to heat-induced epitope retrieval in high pH Target Retrieval Solution (Dako, Carpinteria, Calif). The slides were placed in peroxidase-blocking solution (Dako) to inhibit non–specific-binding activity.
Immunostaining for E-cadherin and EMT-related proteins, including Slug, Twist and Zeb1, was performed by placing the sections in a microwave oven for 30 minutes for antigen retrieval. After primary antibody treatment, sections were examined using the EnVision HRP detection system (Dako). The antigen-antibody complex was visualized with DAB+ liquid chromogen (Dako) and counterstained with hematoxylin before mounting. Representative histologic findings and the immunoreactivity of E-cadherin and EMT markers in APC are shown in Figure1 .
Immunostaining for E-cadherin and EMT-related proteins, including Slug, Twist and Zeb1, was performed by placing the sections in a microwave oven for 30 minutes for antigen retrieval. After primary antibody treatment, sections were examined using the EnVision HRP detection system (Dako). The antigen-antibody complex was visualized with DAB+ liquid chromogen (Dako) and counterstained with hematoxylin before mounting. Representative histologic findings and the immunoreactivity of E-cadherin and EMT markers in APC are shown in Figure
3
Alkaline Comet Assay for DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The alkaline comet assay was performed as described previously [22] . In brief, cells were incubated with H2O2 for 30 min on ice followed by the incubation in their respective culture medium at 37C for the indicated periods of time. After centrifugation, cells were embedded in 0.75% low-gelling temperature agarose (A4018; Sigma) on poly-L-lysine-coated glass slides (Matsunami Glass, Osaka, Japan). Then the cells were lysed by incubating the slides at 4C in lysis buffer [1% Triton X-100, 0.5% N-Lauroryl sarcosine sodium salt, 2.5 M NaCl, 100 mM EDTA (pH 8.0), and 10 mM Tris-HCl (pH 10.0)]. After washing twice in PBS, the slides were immersed in pre-chilled electrophoresis buffer (0.3 M NaOH and 1 mM EDTA in water) for 40 min and electrophoresis was performed at 25 V for 50 min at 4C. Then the samples were washed twice in 400 mM Tris-HCl (pH 7.0) at room temperature and soaked in ethanol for 5 min, followed by overnight drying at 37C. DNA was detected by ethidium bromide staining for 30 min at room temperature. After rinsing in PBS, slides were observed via confocal microscopy and quantified using the Metamorph software.
4
Spinal Cord Tissue Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 28 dpi, mice that were deeply anaesthetised using isoflurane were perfused with 50 mL of phosphate-buffered saline (PBS) followed by 50 mL of 4% paraformaldehyde. The isolated spinal cord was fixed with 4% paraformaldehyde in PBS for 2 h at 4 °C, and then immersed in 30% sucrose for 48 h. Spinal cords were cut into 5 mm segments centred on the injury site before being frozen in the presence of optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Serial longitudinal sections (20 μm thick) or cross sections (10 μm thick) of the cord were prepared through the lesion site from frozen tissues at −25 °C and mounted on poly-L-lysine-coated glass slides (Matsunami, Osaka, Japan).
5
Immunohistochemical Localization of Leptin Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections of human buccal mucosa and rabbit gingiva were transferred onto poly-l -lysine-coated glass slides (Matsunami Glass, Osaka, Japan). After deparaffinization with xylene and rehydration with descending concentrations of ethanol, endogenous peroxidase was blocked by treatment with 3% H2O2 in methanol for 1 hour at room temperature (RT). After treatment with 10% normal rabbit serum at RT for 10 min, sections were incubated with the primary antibodies (goat polyclonal antibody against Ob-R, Santa Cruz Biotechnology, Inc., CA, USA) diluted 1∶500 in PBS (pH 7.4) containing 1% bovine serum albumin at 4°C overnight. After washing with PBS, the localization of Ob-R was visualized using a Histofine SAB-PO (G) kit (Nichirei Corporation, Tokyo, Japan) and a 3,3′-diaminobenzidine (DAB) substrate kit (Nichirei). Sections were counterstained with hematoxylin and mounted. The specificity of the immunoreaction was confirmed by incubation with normal goat IgG and normal goat serum instead of the primary antibodies.
6
Fluorescent Immunostaining for Cell Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent immunostaining was performed by attaching, fixing, and staining cells on poly-l-lysine-coated glass slides (Matsunami Glass, Osaka, Japan), with imaging and quantification performed with a Cellomics ArrayScan VTI HCS Reader (ThermoScientific), using methods described previously20 (link),28 (link).
7
Fluorescent Immunostaining for Cell Imaging
8
Histological Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed in 10% formalin at room temperature and embedded in paraffin wax. Sections of 3 µm thickness were cut from the paraffin block and mounted on poly-L-lysine-coated glass slides (Matsunami Glass Ind., Ltd., Tokyo, Japan). Paraffin sections were dewaxed at 37 °C for 7 h and rehydrated using a 70–100% alcohol gradient (70%; 90%; 95%; and three times 100%), 90 min for each interval, as previously described21 (link). Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to observe changes in the alveolar septum and injury and accumulation of inflammatory factors in the alveolar cavity using a light microscope (Olympus BX50, Olympus, Tokyo, Japan).
9
Spinal Cord Injury Histological Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed 1 week (n = 3/group), 3 weeks (n = 3/group), and 7 weeks (after the termination of locomotor assessment, n = 10/group) after the transplantation. The animals were deeply anesthetized with intraperitoneal sodium pentobarbital (Kyoritsu Seiyaku, Tokyo, Japan) and perfused transcardially with 4% paraformaldehyde (Wako) in phosphate-buffered saline (PBS, pH 7.4; Life Technologies Japan, Tokyo, Japan). Spinal cords including the lesion site were removed and postfixed in the same fixative for 24 h. Then tissues were immersed in 20% sucrose in PBS at 4°C. After that, the tissue was embedded in OCT compound (Tissue-Tek, Sakura Finetech. Tokyo, Japan), frozen on dry ice, and sectioned on a cryostat. The sagittal serial frozen spinal cord sections (25 μm in thickness) were mounted onto poly-llysine-coated glass slides (Matsunami, Tokyo, Japan). Each slide contained five sliced sections at 100-μm intervals, and the sections for each slide were offset by 25 μm from the previous slide in the set. In this way, we were able to cover the central 500 μm of the lesion at 25-μm intervals in five slides.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!