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 secondary antibody dilution buffer
Manufactured by Beyotime
QuickBlock™ Secondary Antibody Dilution Buffer is a ready-to-use solution designed to dilute secondary antibodies for immunoassays. It contains a balanced formulation of salts, buffers, and stabilizers to maintain antibody activity and specificity.
Automatically generated - may contain errors
Lab products found in correlation
Quickblock primary 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)
Ab178847, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
P0102, by Beyotime (1 mentions)
Ab112, by Abcam (1 mentions)
Ecl plus detection kit, by Beyotime (1 mentions)
Quickblock buffer, by Beyotime (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
3 protocols using quickblock secondary antibody dilution buffer
1
Immunofluorescent Labeling of Neural Markers
2
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.
3
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.
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