Immunofluorescent reactions were performed on free-floating sections following previously published methods 3 (link). Briefly, sections were preincubated with QuickBlock Blocking Buffer (P0260; Beyotime, Shanghai, China) for 1 h. After washing in PBS (3×10 min), the sections were incubated overnight at 4°C with primary antibodies diluted in QuickBlock Primary Antibody Dilution Buffer (P0262; Beyotime). The primary antibodies used in this study were mouse anti-AnkyrinG (1 : 1000, MABN466; Merck Millipore, Shanghai, China), rabbit anti-NeuN (1 : 3000, ab177487; Abcam, Shanghai, China), and rabbit anti-Nav1.6 (1 : 1000, ASC-009; Alomone Labs, Jerusalem, Israel). After several washes in PBS, sections were incubated with the secondary antibodies in QuickBlock Secondary Antibody Dilution Buffer (P0265; Beyotime) for 2 h at room temperature. The secondary antibodies were goat anti-Rabbit IgG H&L (Alexa Fluor 488) (1 : 1000, ab150077; Abcam) and goat anti-Mouse IgG H&L (Alexa Fluor 555) (1 : 1000, ab150114; Abcam). After secondary antibody incubation and several washes in PBS, sections were mounted on clean glass slides with glycerol and sealed with nail polish. Control sections were labeled simultaneously using the same procedure as described above, with the exception that the primary antibody was substituted with QuickBlock Primary Antibody Dilution Buffer.
Quickblock primary antibody dilution buffer
Manufactured by Beyotime
Sourced in China
QuickBlock™ Primary Antibody Dilution Buffer is a ready-to-use solution designed to dilute primary antibodies for immunoassays. It maintains the stability and activity of the antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Quickblock secondary antibody dilution buffer, by Beyotime (3 mentions)
Ab177487, by Abcam (1 mentions)
Ab150114, by Abcam (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Ab150077, by Abcam (1 mentions)
Asc 009, by Alomone (1 mentions)
Ab227978, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Df7997, by Affinity Biosciences (1 mentions)
Beyoecl moon, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab178847, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
P0102, by Beyotime (1 mentions)
Ab112, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Amersham hybond, by GE Healthcare (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Ripa reagent, by Merck Group (1 mentions)
11 protocols using quickblock primary antibody dilution buffer
1
Immunofluorescent Labeling of Neural Markers
2
Knockdown of ENO1 in FLS Cells
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According to the GenBank database, the siRNA sequence(Table S2 ) of rat and human ENO1 gene designed and obtained from JTSBIO Co,Ltd (Wuhan, China). ENO1-siRNA and negative control (NC) vectors were transfected into primary cultured FLS cells of rat and human.
The knees of rat models were embedded in paraffin, sectioned at a thickness of 5-μm, and then stained with hematoxylin and eosin. Cells were lysed using RIPA buffer (Beyotime, China). Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight. Membrane was subsequently incubated with HRP-conjugated secondary antibodies. BeyoECL Moon (Beyotime, China) was used to visualize the protein bands. Detection was performed using an automated chemiluminescence image analysis system (SyngeneG Box chemi xx6, British) and the quantification of the bands was performed using Image J software.
The knees of rat models were embedded in paraffin, sectioned at a thickness of 5-μm, and then stained with hematoxylin and eosin. Cells were lysed using RIPA buffer (Beyotime, China). Protein was electrophoresed with SDS-PAGE gel and transferred to PVDF membrane, blocked using QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China), and then incubated with the ENO1 antibodies (ab227978, Abcam), GRN antibodies (DF7997, Affinity Biosciences), PTGS2 antibodies (K001561P, Solarbio) and ACSL4 (ab155282, Abcam) at 4°C overnight. Membrane was subsequently incubated with HRP-conjugated secondary antibodies. BeyoECL Moon (Beyotime, China) was used to visualize the protein bands. Detection was performed using an automated chemiluminescence image analysis system (SyngeneG Box chemi xx6, British) and the quantification of the bands was performed using Image J software.
3
Immunofluorescence Staining of Mouse Midbrain
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The brains tissues of mice were fixed in 4% paraformaldehyde buffered with PBS at 4°C for 24 h, followed by changing into 30% sucrose solution for another 24 h. After embedding by optimum cutting temperature (O.C.T.) compound (Solarbio, Shanghai, China), the midbrain was cut into 16-μm-thick serial brain sections containing the SNpc for the subsequent immunofluorescence staining. The slices were incubated with blocking solution (P0102, Beyotime, Beijing, China) including BSA and Triton X-100 for 1 h followed by the incubation overnight at 4°C with anti-tyrosine hydroxylase (TH, ab112, Abcam, Cambridge, MA, USA, 1:500) and anti-ionized calcium-binding adapter molecule 1 (Iba-1, ab178847, Abcam, 1:200) antibodies, respectively. The primary antibodies were diluted by QuickBlock™ Primary Antibody Dilution Buffer (P0262, Beyotime). Then the slices were incubated with secondary fluorescent antibodies fluorescein isothiocyanate (FITC)-labeled Goat Anti-Rabbit IgG (green, A0562, Beyotime, 1:1,000) for 2 h. The secondary antibodies were diluted by QuickBlock™ Secondary Antibody Dilution Buffer (P0265, Beyotime). After that, fluorescence microscope (Carl Zeiss, Oberkochen, Germany) were applied to photograph.
4
Macrophage Inflammation Regulation Assay
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Macrophage cell lines, RAW264.7 and Ana-1 cells (3 × 105 cells in a 6-well plate) were co-cultured with LPS (1 μg/ml) and TMP (10 μg/ml) or Dex (5 μM) for 24 h. In vivo experiments, the lung tissues were dissolved into lysates in RIPA Lysis Buffer (Beyotime, Shanghai, China) with a Phenylmethanesulfonyl fluoride and phosphatase inhibitor (Beyotime, Shanghai, China). Equal amounts of protein (50 μg/lane) were separated electrophoretically through SDS-PAGE (10%, 12%) and transferred onto a PVDF membrane (Millipore Corp, Billerica, MA, United States). The PVDF membranes were then blocked for 2 h at room temperature with 5% non-fat milk in TBST. The membranes were incubated with appropriate primary antibodies (1:1000) in QuickBlock™ Primary Antibody Dilution Buffer (1:1,000, Beyotime, Shanghai, China) for overnight at 4°C and horseradish peroxidase-conjugated secondary antibodies 1:1,000 with 1% non-fat milk for 2 h at room temperature. Images were digitally acquired with chemiluminescence image analysis system (Tanon, Shanghai, China). The expression levels of target proteins were normalized with β-actin or GAPDH as an internal control.
5
Western Blot Protein Quantification
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The proteins were extracted from tissues by homogenization in RIPA reagent (Sigma), and protein concentrations were determined by BCA Protein Assay Kit (Solarbio, Beijing, China). Proteins from each group were subjected to into analysis. The proteins were resolved by SDS/PAGE followed electrotransfer to PVDF membrane (Amersham Hybond, GE Healthcare Bio-Sciences, Pittsburgh, PA, United States). The PVDFs were incubated with the primary antibody in QuickBlock™ primary antibody dilution buffer (P0256, Beyotime, Haimen, China). The next day, remove the primary resistance, the primary antibody was detected with the secondary antibody in PBS containing 0.05% Tween 20, and then chemiluminescenced via ECL (RPN2232, GE Healthcare, United Kingdom). The protein expression was normalized to that of GAPDH as the internal loading using Quantity One software (RRID:SCR_014280, Bio-Rad, CA, United States).
6
Western Blot for DNA Damage Signaling
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Western blotting was conducted to detect the protein expression of DNA damage signaling pathways. Cells pellets were lysed by RIPA lysis buffer. Then, 20-40 μg protein from each experimental condition was subjected to sodium dodecyl sulfate-polyacrylamide gel for electrophoresis, then transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with QuickBlock™ Primary Antibody Dilution Buffer (Beyotime, China) for 10 min at room temperature and then incubated with the appropriate primary antibody overnight at 4°C. Primary antibodies were diluted in Primary Antibody Dilution Buffer (Beyotime, China). The membranes were washed with TBS-T, followed by probing with species-specific secondary antibodies conjugated with HRP, diluted in Secondary Antibody Dilution Buffer (Beyotime, China). Protein bands were detected using Enhanced Chemiluminescence Substrate (PerkinElmer, USA).
7
Western Blot Analysis of E. coli FliC
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E. coli cells of two groups, RP437-TC-FliC and RP437-TC-FliC carrying plasmid pBAD18-TC-FliC supplemented with 0.2% arabinose, were grown in LB (the latter containing ampicillin) at 30 °C for 5 h. Afterwards, the cultures were heated at 65 °C for 5 min and harvested by centrifugation. The obtained cell pellets were then resuspended in Western Sample Buffer (950 μl 2 × Laemmli Buffer, Bio-Rad; 50 μl 2-Mercaptoethanol, Sigma) and heated at 95 °C for 10 min. Samples were normalized by cell density. After separated by SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad), samples were blocked in QuickBlock™ Buffer (Beyotime) for 1 h at room temperature. Primary incubation was with an anti-FliC rabbit antibody (Abcam, ab93713) diluted 1:10,000 or an anti-MreB rabbit antibody (a gift from Zhen Liu) diluted 1:5000 in QuickBlock™ Primary Antibody Dilution Buffer (Beyotime) overnight at 4 °C. Secondary incubation was with goat anti-rabbit HRP conjugate (Beyotime) diluted 1:2000 in QuickBlock™ Secondary Antibody Dilution Buffer (Beyotime) for 1 h at room temperature. Blots were treated with an ECL plus detection kit (Beyotime). Three independent experiments were performed.
8
Western Blot Analysis of Artemin
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Cells were harvested, washed with phosphate buffered saline (PBS) twice and incubated at 98°C in radioimmunoprecipitation assay lysis buffer (Beyotime, Beijing, China). The protein extracts were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with Quickblock Primary Antibody Dilution Buffer (Beyotime, Beijing, China) for one hour and then incubated with anti‐artemin rabbit polyclonal antibody (AB178434, ABCAM) or anti‐GAPDH antibody (KM9002T, Sungene, Tianjin, China) as a control.
9
Immunostaining of Frozen Bladder Tissue
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All antibodies applied in the current assays are listed in Table S6 . Briefly, frozen bladder tissue sections were placed directly on slides and then fixed with immunostaining fixative (Beyotime, #P0098, China). Then, the slides were washed twice with immunostaining washing solution (Beyotime, #P0106, China) for 5 min each. The immunostaining blocking solution (Beyotime, #P0102, China) was added and blocked overnight at 4 °C. The primary antibody was diluted to the appropriate ratio according to the instructions with QuickBlock Primary antibody dilution buffer (Beyotime, #P0262, China) and then added to the sections and incubated overnight at 4 °C with slow shaking. immunostaining washing solution was then added and washed slowly by shaking on a side shaker for 5 min; this was repeated three times. Based on the instruction manual of the secondary antibody, dilution with the secondary antibody buffer for immunofluorescence (Beyotime, #P0106, China) or with secondary antibody solution for immunohistochemistry (Beyotime, #P0110, China) was performed. immunostaining washing solution was added and washed slowly by shaking on a side shaker for 5 min, which was repeated three times. The results were observed using the appropriate tools.
10
Western Blot Analysis of Infected Cells
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The uninfected and infected cells were seeded after 48 hpi, washed three times with PBS, and lysed in RIPA buffer (20 mM Tris, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.5% Triton-X, 0.1% SDS) with a protease inhibitor cocktail (P1050, Beyotime, China) and phosphatase inhibitor cocktail (P1050, Beyotime, China). Cell lysates were centrifuged for 15 min at 12 000 g at 4 °C to sediment cell debris after 10 min incubation on ice. The total protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Roche). Membranes were blocked with 5% (w/v) nonfat dry milk (#9999, Cell Signalling Technology, Danvers, MA, USA) for 1 h at 37 °C, and incubated overnight at 4 °C with the primary antibody diluted in QuickBlock™ Primary Antibody Dilution Buffer (P0256, Beyotime, China), following three washes with TBST (Tris-buffered saline containing 0.05% Tween 20), and then 1 h at RT with the secondary antibody diluted in PBS. After three washes with TBST, the membrane was exposed with the ECL Western blot detection kit (#34580, Thermo Fisher Scientific) and imaged using an Azure Biosystems C300 imaging system. The relative optical density of Western blot bands was measured by ImageJ, values of which were analyzed using the GraphPad Prism for the t-test.
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