For Iba-1 staining, 3–4 slices per mouse spanning the olfactory bulb were permeabilized and blocked with a solution containing 0.1% Triton X-100 (Fisher), and 5% goat serum in PBS for 1 hr at room temperature or blocked with Starting Block (ThermoFisher) with 0.3% TritonX-100 for 1 hr at room temperature and then incubated overnight at 4°C with the primary antibodies rabbit anti-Iba-1 (Wako: 019–19741, RRID:AB_839504 ) at 1:500 or rabbit anti-GFAP (Dako: Z0334, RRID: AB_10013382 ) at 1:1000 and then secondary antibodies (Alexa goat-647 anti-Rabbit) for 2 hr at room temperature. For BrdU/NeuN staining, one of every eight slices per mouse was chosen. Slices were washed in PBS with 0.1% Triton X three times for five minutes each before being incubated for in 2N HCl for 10 min at room temperature and then 20 min at 37°C. They were then placed in 0.1M Boric Acid buffer for 15 min and washed again three times with PBS. All slices were then incubated in Starting Block (ThermoFisher) with 0.3% Triton X for one hour before being staining in in PBS with 0.3% Triton X with rat anti-BrdU (Abcam: 6326 at 1:200), and mouse anti-NeuN (Millipore: MAB377 at 1:200) primary antibodies for 36–48 hr at 4°C and then secondary antibodies (Alexa Fluor 488 and 594 at 1:200) for 2 hr at room temperature. Slices were treated with 0.2% w/v Sudan Black in 70% EtOH for 5 min before mounting.
Startingblock
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
StartingBlock is a laboratory equipment designed for sample preparation. It provides a stable platform for holding various types of sample tubes or containers during sample preparation workflows. The core function of StartingBlock is to secure and organize samples in a standardized manner to facilitate efficient and consistent sample handling.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (3 mentions)
Proteinase and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Immobilon fl pvdf membrane, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Ox40 fc, by Bio-Techne (2 mentions)
Versamax, by Molecular Devices (1 mentions)
Sc 65755, by Santa Cruz Biotechnology (1 mentions)
Sc 1616 hrp, by Santa Cruz Biotechnology (1 mentions)
Ultra tmb, by Thermo Fisher Scientific (1 mentions)
Spectramax spectrophotometer, by Molecular Devices (1 mentions)
Anti alix, by Cell Signaling Technology (1 mentions)
Anti calnexin, by Abcam (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Anti cd9, by System Biosciences (1 mentions)
Phosphatase inhibitor, by Boster Bio (1 mentions)
49 protocols using startingblock
1
Immunohistochemical Staining of Brain Slices
2
Protein Analysis of Termite and Cockroach
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Termite and cockroach protein samples (25 μg/lane) were resolved on Novex 4–20% Tris-Glycine or 4–12% Bis-Tris gels (Life Technologies). NuPAGE LDS sample buffer (Life Technologies) was added in a 4:1 v/v ratio. Proteins were visualized using Safe Stain (Life Technologies, Carlsbad, CA, USA) and gel images were visualized using an Odyssey CLX infrared imaging system (LI-COR, Lincoln, NE, USA). Immunoblots using rabbit and mouse full-length antibodies were performed as described in Mattison et al., 2014 [27 (link)], except secondary antibodies were IRdye800 labeled donkey anti-rabbit and donkey anti-mouse antibodies (LI-COR). Immunoblots using IRdyes were visualized using an Odyssey CLX infrared imaging system (LI-COR, Lincoln, NE, USA). For immunoblots using scFv antibody fragments, proteins separated by SDS-PAGE were transferred onto PVDF membranes (0.2 μM, Life Technologies). After blocking with Starting Block (Life Technologies), the membrane strips were incubated with scFv (1 μg/mL diluted in Starting Block). The binding efficiency of scFvs was detected using HRP conjugated anti-HA antibody (1:2000 in PBS-T + 3% BSA). The HRP signal was quantified using a Densitometer G Box from Syngene (Frederick, MD).
3
Serological ELISA Protocol for Staphylococcal Toxins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serology ELISAs were performed as described previously. Briefly, 96-well plates were coated with 300 ng/well of wild type (WT) proteins Hla, LukS, LukF, LukD, LukE, HlgA, HlgB, or HlgC or 100 ng/well of WT SEA, SEB, SEC1-3, SED, SEE, SHE, SEI, SEJ, TSST-1, or SpeB overnight at 4°C. Plates were washed and blocked with Starting Block (Thermo Fisher) for 1 h at room temperature (RT) followed by three washes. Plates were incubated for 1 h at RT with the test serum samples (diluted semi-log) and washed three times before applying goat anti-monkey IgG (H&L)-HRP (Horse Radish Peroxidase) at 0.1 µg/ml in Starting Block buffer. Plates were incubated for 1 h at RT, washed, and incubated with TMB (3,3′,5,5′-tetramethylbenzidine) to detect HRP activity for 30 min. 50 µl of stop solution (2N H2SO4) was added to all the wells. Optical density at 450nm was measured using a Versamax™ plate reader (Molecular Devices, CA). Data analysis for full dilution curves was performed using the SoftmaxPro software version 5.4.5 (Molecular Devices, CA) and ND50 values extrapolated.
4
Detecting Proteasome Subunit in Fly Lysate
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot samples were prepared as described25 (link). Briefly, tissue samples stored at −80 °C were thawed in RIPA buffer (200 μL to 400 μL) supplemented with proteinase- and phosphatase-inhibitor cocktail (Sigma), then homogenized with an electrical homogenizer followed by short sonication to form a homogeneous tissue lysate. The lysate solution was centrifuged at 18,000 × g for 30 min at 4 °C. Protein concentration was measured with the BCA assay (Thermo Fisher). Cell lysate of fallopian tube cells FT237 and FT246 were obtained from Dr. Analisa Difeo (University of Michigan, MI). Thirty to forty μg protein per sample was subjected to SDS-PAGE analysis. The lysates of ovarian surface epithelial cells and fallopian tube cell 15–20 µg protein was used for WB analysis. Proteins were then electro-transferred to methanol activated immobilon-FL PVDF membranes (EMD Millipore). Membranes were blocked with Starting Block (Thermo Fisher) for 1 hr at room temperature and incubated with primary antibodies overnight at 4 °C. Dylight 800-conjugated secondary antibodies were used for detection (Thermo Fisher, 1:5000, 5% milk, 1 hour, RT) of fluorescent signals in Odyssey Imaging Systems (LI-COR Biosciences).
6
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples stored at -80 °C were thawed in RIPA buffer (200 μL to 400 μL) supplemented with proteinase- and phosphatase-inhibitor cocktail (Sigma), then homogenized with an electrical homogenizer followed by short sonication to form a homogeneous tissue lysate. The lysate solution was centrifuged at 18,000 x g for 30 min at 4 °C. Protein concentration was measured with BCA assay (Thermo Fisher). Thirty to forty μg proteins per sample was subjected to SDS-PAGE analysis. Proteins were then electro-transferred to methanol activated immobilon-FL PVDF membranes (EMD Millipore). Membranes were blocked with starting block (Thermo Fisher) for 1 hr at room temperature and incubated with primary antibodies overnight at 4 °C. Dylight 800-conjugated secondary antibodies were used for detection (Thermo Fisher, 1:5000, 5% milk, 1 hour, RT) of fluorescent signal in Odyssey Imaging Systems (LI-COR Biosciences).
7
ELISA Protocols for Vaccine and T3SP Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ELISAs were performed as previously described with slight modifications18 (link). Briefly, for vaccine-strain specific ELISA, formalin-killed vaccine strain was coated to high binding ELISA plates at an equivalent of 108 cells per well at 4 °C, overnight in bicarbonate buffer (pH of 9.6). For T3SP ELISA, EspA and EspB were coated at 10 µg/mL20 (link) at 4 °C, overnight in bicarbonate buffer (pH of 9.6). Coated ELISA plates were washed 5 times with PBS + 0.05% Tween-20 and wells were blocked with StartingBlock (Thermo Fisher Scientific) for 1 h at room temperature. Serum or fecal samples were analyzed in duplicate at three, ten-fold serial dilutions starting at 1:100. Plates were washed 5 times with PBS + 0.05% Tween-20 and Anti-bovine IgA-HRP, IgM-HRP (1:10,000 dilutions) or IgG-HRP, IgG1-HRP, IgG2-HRP (1:50,000 dilution) (Bethyl Labs) were added to plate wells as per manufacturer’s instructions. Ultra-TMB (Thermo Fisher Scientific) was added to plates after 10 washes with PBS + 0.5% Tween-20. Plates were allowed to develop for approximately 5 minutes and stopped with 1M HCl. Plates were immediately analyzed for absorbance at 450 nm (OD450) on a SpectraMax Spectrophotometer (Molecular Devices, Sunnyvale CA).
8
Exosome Protein Characterization by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and exosomes were lysed in RIPA buffer (Cell Signaling). Sample loading was normalized according to BCA protein assay kit (Pierce) and proteins were separated following an electrophoretic gradient across polyacrylamide gels. Wet electrophoretic transfer was used to transfer the proteins in the gel onto 0.45 µm pore-size PVDF membranes (Novex). The protein blot was blocked for 1 h at room temperature with starting block (Thermo) and incubated overnight at 4 °C with the following primary antibodies: anti-Calnexin (Abcam, ab22595), Anti-Alix (Cell Signaling, 3A9), anti-TSG-101 (Abcam, ab83), anti-CD9 (SBI, ExoAb-kit-1). All antibodies were diluted 1:1000. Afterwards, horseradish peroxidase (HRP)-conjugated secondary antibodies (EMD Millipore, 12-348 and 12-349, 1:5000) were incubated for 40 min at room temperature. Washes after antibody incubations were done on an orbital shaker, three times at 10-min intervals, with TBS-Tween20. Blots were developed with the Dura and Pico supersignal chemiluminescent reagents (Thermo). Uncropped versions of all western blot images are presented at the end of the Supplementary Information accompanying this article.
9
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethanesulfonylfluoride (BOSTER, Shenzhen, China) and phosphatase inhibitors (BOSTER, China) was used to extract cells. Following the determination of the protein concentrations using the BCA protein assay kit (Beyotime, China), 50 µg of protein extract was separated through gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). After being blocked with StartingBlock (Thermo, Waltham, MA, USA) for 15 min, the membranes were incubated overnight at 4 °C with primary antibodies such as Sirt3 (1:1000), SOD2 (1:1000), and GAPDH (1:5000). Subsequently, the membranes underwent rinsing and were incubated for 2 h with horseradish peroxidase-labeled secondary antibodies (Solarbio, Beijing, China). Following rinsing, the protein signals were visualized using Image Lab 3.0 software.
10
SARS-CoV-2 Antibody-Mediated Complement Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microtiter plates were coated and washed as described above and blocked with Starting Block (ThermoFisher) for 10 min. Test serum was heat inactivated at 56°C for 30 minutes, before being diluted 1 in 5 with Starting Block supplemented with 5 mM calcium chloride and 5 mM magnesium chloride. 50 µl was added to the antigen-coated plate and incubated for 1hr at 37°C. After washing, 50 µl COVID negative normal human serum (same source used throughout all assays, containing no detectable S or N specific antibodies as measured by IgGAM ELISA) at a dilution of 1:40 (in 2% Starting Block plus 5 mM calcium chloride and 5 mM magnesium chloride) was added to each well for 1 hr at 37°C. The following anti-human monoclonal complement antibodies (100ul, diluted in PBS-0.1% Tween 20) were added and incubated at 37°C for 1 hr: mouse anti-C4b, 1:22,500 (Invitrogen, LF-MA0198); mouse anti-C3b, 1:10,000 (Invitrogen MA1-70053); mouse anti-C5b, 1:10,000 (Invitrogen DIA 011-01-02). HRP conjugated goat anti-mouse at a 1:4000 (Southern Biotech 1010-05) was then incubated at RT for 1 h. Plates were developed and read as described above.
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!