We fixed and permeablized astrocytes with 4% paraformaldehyde and 0.2% Triton‐X100 in phosphate buffered saline (PBS). After blocking in 10% donkey serum, we stained astrocytes with the following primary antibodies: anti‐GFAP (1:1,500. Biolegend, 829401), Ki67 (1:200. Invitrogen MA5‐14520), enhanced green fluorescent protein (EGFP) (1:1,000. Aves Labs GFP‐1020), mCherry (1:600. Clontech 632543), Sox9 (1:2,000. Millipore AB5535), Bromodesoxyuridine (BrdU) (1:500, Novusbio NB500‐169), and fluorescent secondary antibodies (Invitrogen). For BrdU staining, cells were pretreated by 2N hydrochloric acid for 20 min before blocking. After three washes in PBS, we mounted the coverslips with VectorShield with DAPI (Vector Labs H1200) and imaged them using an Evos FL Auto 2 inverted fluorescence microscope (Invitrogen) with ×10, and ×20 lenses and the Imager M2 upright fluorescence microscope (Zeiss) with ×10, ×20, and ×40 lenses. We cropped the images with the FIJI and Photoshop softwares.
Ma5 14520
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MA5-14520 is a laboratory scale designed for precise weighing applications. It features a high-precision balance with a capacity of up to 520 grams and a readability of 0.01 grams.
Automatically generated - may contain errors
Lab products found in correlation
Nb500 169, by Thermo Fisher Scientific (2 mentions)
Evos fl auto 2 inverted fluorescence microscope, by Thermo Fisher Scientific (2 mentions)
Imager m2 upright fluorescence microscope, by Zeiss (2 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (2 mentions)
Gfp 1020, by Aves Labs (2 mentions)
A32766, by Thermo Fisher Scientific (2 mentions)
Ab2302, by Abcam (2 mentions)
Re7116 ce, by Leica (2 mentions)
Ab5535, by Novus Biologicals (1 mentions)
Vectorshield with dapi, by Vector Laboratories (1 mentions)
Sh800, by Sony (1 mentions)
Pa5 106697, by Thermo Fisher Scientific (1 mentions)
Ab182422, by Abcam (1 mentions)
Ab5690, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Ab209775, by Abcam (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Alexa 594, by Jackson ImmunoResearch (1 mentions)
Alexa 488, by Jackson ImmunoResearch (1 mentions)
42 protocols using ma5 14520
1
Immunocytochemical Analysis of Astrocytes
2
Single-cell Analysis of Testicular Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were prepared from testes from E13.5 and E14.5 embryos by incubation with 0.15% trypsin-EDTA at 37°C for 5 min, and then washed with DMEM containing 0.5% BSA (wash medium). After discarding the supernatant, cells were fixed in 100 μl of 4% PFA for 10 min at RT, followed by the addition of 900 μl of cold 100% EtOH and incubated at 4°C overnight. After spinning down for 5 min at 400 × g, cells were washed with wash medium and reacted with primary antibodies for 1 h at RT at the following dilutions: mouse anti-NANOS2 (1:200, (Suzuki et al., 2007 (link))), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520) and rat anti-GCNA (1:4,000, a gift from Y. Nishimune, Osaka University). Then, cells were washed with wash medium and reacted with secondary antibodies labeled with Alexa Fluor 488, 594 or 647 (1:1,000, Invitrogen A32766, A21209 and A32795) for 30 min at RT. Next, washed cells were re-suspended in 0.1% BSA-containing PBS and analyzed by a JSAN Desktop Cell Sorter or SH800 (SONY). The data were visualized using flowCore package (Ellis et al., 2019 ).
3
In vivo Evaluation of NUSAP1 on Tumor Growth
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All mice experiments were conducted in accordance with the protocols approved by the Ethical Committee for Animal Experimentation of the Army Medical University. SPF grade female nude mice (5 weeks old with a mean body weight of 20 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). To evaluate the effect of NUSAP1 on the cell proliferation ability in vivo, 1 × 107 control or NUSAP1-knockdown A549 and MCF-7 cells were subcutaneously inoculated in the underarm region of the right foreleg of each mouse (5 mice for per group). The tumor diameter and weight of tumor-bearing mice were measured at 3, 6, 9, 12, 16, 20, and 22 days respectively after inoculated. Tumor volume was calculated using the formula: volume = (long diameter) × (short diameter)2/2. After 20 days, the mice were euthanized, and the tumors were isolated, weighted, photographed. The tumors were then lysed and total protein was extracted for western blotting analysis to assess the levels of NUSAP1 and KI67 proteins in each tumor. Antibodies used for western blotting was rabbit anti-NUSAP1 (PA5-106697, Invitrogen) at a 1:1000 dilution, rabbit anti-KI67 (MA5-14520, Invitrogen) at a 1:1000 dilution, and mouse anti-β-Actin (3700S, CST, MA, USA) at a 1:20000 dilution.
4
Immunohistochemical Analysis of Tumor Infiltrates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The harvested tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry (IHC) staining was performed with the following primary antibodies: anti-CD3 (1:100, ab5690, Abcam, Cambridge, UK), anti-CD4 (1:100, ab183685, Abcam), anti-CD8 (1:100, ab209775, Abcam), F4/80 (1:50, 14-4801-81, Invitrogen, San Diego, CA, USA), anti-CD163 (1:200, ab182422, Abcam), and anti-Ki67 (1:200, MA5-14520, Invitrogen).
5
Multimarker Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staining for FOXA2 (1:300, WRAB-FOXA2, Seven Hills Bioreagents), E-cadherin (1:300, 3195s, Cell Signaling Technology), EGFR (1:100, 4267, Cell Signaling Technology), Ki67 (1:200, MA5-14520, Invitrogen), and CK8 (1:100, TROMA-1, Hybridoma Bank, Iowa) was performed using secondary antibodies conjugated with Alexa 488 or Alexa 594 (1:300, Jackson Immuno Research). Nuclear staining was performed using Hoechst 33342 (4 µg/ml, H1399, Thermo Scientific). Tissue sections from control and experimental groups were processed on the same slide for each experiment. Images presented are representative of three independent experiments.
6
Immunohistochemical and Immunofluorescent Staining
7
Immunohistochemical Analysis of Cell Proliferation and Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining was performed according to the manufacturer's guidelines (Finetest, IHC0007). Briefly, 4-μm thick paraffin-embedded tissue blocks were sectioned onto slides, and the sections were deparaffinized. The sections were then blocked for 1 h with blocking serum at 37 °C. The slides were incubated with primary antibodies against Ki-67 (Invitrogen, MA5-14520) and caspase-3 (Invitrogen, PA1-29157) overnight at 4 °C, washed, and incubated using poly-HRP goat anti-rabbit IgG as a secondary antibody at room temperature for 1 h. Color was developed using 3,3'-diaminobenzidine tetrahydrochloride for 2–10 min, and the sections were counterstained with hematoxylin. Images were acquired using a Nikon microscope (Tokyo, Japan) and digitally processed using ImageJ software to measure the percentage of cells exhibiting positive staining of Ki-67 and caspase-3.
8
Immunofluorescence Staining of Astrocytes
9
Immunostaining of Embryonic Gonad Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryonic gonads were fixed in 4% PFA for 30 min at 4°C. Gonads were next submerged in 10 and 20% sucrose in PBS for 1 h each at 4°C, and in 30% sucrose in PBS overnight at 4°C. The gonads were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in liquid nitrogen. Six-micrometer-thick sections of each gonad were applied to glass slides and autoclaved in TRS (Dako). After pre-incubation with 3% skim milk in PBS-T (PBS with 0.1% Tween20 (Sigma-Aldrich)) at RT for 30 min, the sections were reacted with primary antibodies overnight at 4°C at the following dilutions: 1:200 for mouse anti-phospho-histone H3 (ser10) (1:200, Sigma-Aldrich, 06-570), goat anti-CDH1 (1μg/ml, R&D, AF748), rabbit anti-DNMT3L (1:500, provided by Dr. Yamanaka), rabbit anti-STRA8 (1:200, abcam #ab49602), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520), rat anti-Ki67 (1:100, Invitrogen AB_10854564) and anti-pS6 (1:200, CST #2211). Secondary antibodies labeled with Alexa 488, 594 or 647 (1:1,000, Invitrogen A21207, A21208, A21209, A32766, A32814, A32754, A32744 and A32795) were used. DNA was counter-stained with DAPI (100 ng/ml). Fluorescence microscopy was performed using Olympus FV1200 and images were processed with ImageJ/Fiji (Schindelin et al., 2012 (link)).
10
Immunohistochemical Analysis of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were embedded in a paraffin block for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Tumor tissue was sectioned in 6 μm slices using Leica Ultracut-R Microtome. We performed IHC using the following antibodies: Ki67, which was used as a marker of proliferation to assess the proliferative activity of the tumor and to determine tumor response to our treatment protocols (Ki-67 recombinant rabbit monoclonal antibody (SP6), Invitrogen #MA5-14520) [25 (link)], and caspase 3 (Cleaved Caspase-3 (Asp175) Antibody #9661, Cell Signaling) [26 (link),27 (link)], which is an important component of apoptosis (Supplementary Methods, Data S1 ).
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!