Independent evaluation of all renal biopsies was performed by two renal pathologists, who were blinded to data assessment and data analysis. Within each renal biopsy specimen, each glomerulus was separately evaluated for present crescents, global sclerosis, and necrosis. The percentage of glomeruli with any of these pathologies was calculated as a fraction of the total number of glomeruli in each renal biopsy. Renal biopsies were scored in accordance with the current version of the Banff score for allograft pathology, as recently described [47 (link),48 (link)]. Kidney sections (formalin-fixed, paraffin-embedded) were deparaffinized in xylene and then rehydrated in ethanol containing distilled water. Utilizing antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), tissue sections were stained. According to the manufacturer’s protocol, labeling was conducted using the NovolinkTM Polymer Detection System (Leica Biosystems, Wetzlar, Germany). Nuclear counterstaining was performed using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules were evaluated for presence, as recently described [12 (link),41 (link)].
A0062
Manufactured by Agilent Technologies
Sourced in United States
The A0062 is an analytical instrument designed for chromatographic separation and detection. It features a high-precision flow control system and a sensitive detection module for reliable quantitative analysis. The core function of this product is to provide accurate and reproducible results for a variety of industrial and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Novolink polymer detection system, by Leica (2 mentions)
Mayer s hematoxylin solution, by Merck Group (2 mentions)
A0423, by Agilent Technologies (1 mentions)
P0049, by Agilent Technologies (1 mentions)
Fast p nitrophenyl phosphate tablets, by Merck Group (1 mentions)
Alkaline phosphatase labeled goat anti rabbit igg antibody, by Merck Group (1 mentions)
Infinite m200, by Tecan (1 mentions)
A0136, by Agilent Technologies (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
A0063, by Agilent Technologies (1 mentions)
Glass coverplates, by Thermo Fisher Scientific (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
Smi 31r, by Fortrea (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
10 protocols using a0062
1
Renal Biopsy Evaluation and Scoring
2
Immunohistochemical Analysis of Kidney Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed, paraffin-embedded kidney sections were deparaffinized in xylene and rehydrated in ethanol containing distilled water. Tissue sections were stained using antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), and labeling was performed using the NovolinkTM Polymer Detection System (Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s protocol. Nuclear counterstaining was performed by using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). As previously described, kidney biopsies were evaluated for the presence/absence of C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules [49 (link),50 (link)].
3
Histochemical Stains for Glomerular Diseases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cases were examined using routine histochemical stains (hematoxylin–eosin (HE), periodic acid-Schiff (PAS), and trichrome stains). Cases in which the diagnosis of diffuse mesangial sclerosis was made were additionally stained by methenamine silver stain and immunohistochemistry for IgA (A0262, Agilent, Santa Clara, CA, USA), IgG (A0423, Agilent), C3 (A0062, Agilent), and C5b-9 (AE11, Quidel, San Diego, CA, USA) to exclude glomerulonephritis. Cases with diffuse mesangial glomerulosclerosis as well as cases showing clear features of thrombotic microangiopathy (TMA) were stained for C4d (BI-RC4D, Biomedica, Vienna, Austria). Morphologic features were only evaluated in areas without autolytic changes. Completely autolytic cases were excluded from the study.
4
Complement Activation Measurement Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LP activity was measured through detection of C3-deposition as described previously [27 (link)], with the following modifications:
Polyclonal rabbit anti-human C3c antibody (A0062; DakoCytomation) was used as the primary antibody in 2000-fold dilution and HRP-conjugated anti-rabbit antibody (A1949, Sigma) was used as the secondary antibody in 40,000-fold dilution.
The signal produced by non-inhibited NHS was considered to be 100% activity. 0% complement activity was defined as the signal produced by 2% NHS treated with 50 μM FUT-175 inhibitor. Results were obtained from four parallel measurements.
Polyclonal rabbit anti-human C3c antibody (A0062; DakoCytomation) was used as the primary antibody in 2000-fold dilution and HRP-conjugated anti-rabbit antibody (A1949, Sigma) was used as the secondary antibody in 40,000-fold dilution.
The signal produced by non-inhibited NHS was considered to be 100% activity. 0% complement activity was defined as the signal produced by 2% NHS treated with 50 μM FUT-175 inhibitor. Results were obtained from four parallel measurements.
5
Salmonella LPS Deposition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assays were performed as in the case of the LP measurements with the following modifications. (i) Plates were coated with 10 μg/ml Salmonella lipopolysaccharide. (ii) Serum dilution buffer lacked CaCl2 and was supplemented with 10 mm EGTA. (iii) SPS was omitted from the buffers. (iv) C3 deposition was measured using polyclonal rabbit anti-human C3c antibody (A0062, DakoCytomation) in a 5,000-fold dilution. Two parallels were measured for each data point.
6
Quantitative C3 Protein Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Maxisorp microtiter plates (96-well, Invitrogen) were coated with rabbit anti-human C3c (Dako #A0062) overnight, at 4 °C. After coating, plates were blocked with Quench for 1 h at 37 °C, then incubated with 50 µl/well with supernatant or lysate of WT or mutant C3 transfected HEK293 cells, diluted in Quench supplemented with 10 mM EDTA, for 1 h at 37 °C. C3 was detected using goat anti-human C3 antibody (Quidel #A304) and HRP-conjugated rabbit anti-goat immunoglobulins (Igs) (Dako #P0049) for 1 h at RT.
7
Quantifying Cell-Associated C3c Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed cells were washed three times with washing buffer (10 mM Tris-HCl, 140 mM NaCl, 5 mM CaCl2, 0.05% Tween 20, pH 7.4) and incubated with BSA (1% in washing buffer) for 2 h at RT. After three washings, cells were incubated with a rabbit polyclonal anti-human C3c (Dako, A0062) diluted 1:5000 in washing buffer, for 1 h 30 min at RT. After washing, cells were incubated with an alkaline-phosphatase-labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:1000 in washing buffer for 1 h 30 min at RT. Cells were then washed and the assay was developed using substrate solution (Fast p-Nitrophenyl Phosphate tablets, Sigma). The absorption was read at OD 405 nm using the spectrofluorimeter (TECAN plate reader, Infinite M200, Switzerland).
8
Immunohistochemical Analysis of Neuronal Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Postsurgically, the paraffin sections were routinely processed for histopathology. The following antibodies and dilutions were applied on 3‐µm tissue sections mounted on negatively charged glass slides: C1q (Dako, A0136, 1:200), C3c (Dako, A0062, 1:100), C3d (Dako, A0063, 1:400), synaptophysin (Syn; Dako, DAK‐SYNAP, 1:500), SMI31 (Covance, SMI31R, 1:25 000), SMI32 (Sternberger, 1:1000), vimentin (Dako, V9, 1:50), and Map2 (Millipore, AP20, 1:8000). Tissue was pretreated with either proteinase K (C1q, C3c, C3d) or pH6 (Syn, Map2, SMI32, SMI31). As a detection system, the EnVision FLEX+ kit (Dako) was utilized and diaminobenzidine as chromogen, executed either with an autostainer (Dako) or via cover plates (Thermo Fisher Scientific glass cover plates). The paraffin sections were counterstained with hematoxylin.
9
Quantitative Complement Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CP activity was measured through detection of C3-deposition as described previously [27 (link)], with the following modifications:
Polyclonal rabbit anti-human C3c antibody (A0062; DakoCytomation) was used as the primary antibody in 2000-fold dilution and HRP-conjugated anti-rabbit antibody (A1949, Sigma) was used as the secondary antibody in 40,000-fold dilution.
The signal produced by non-inhibited NHS was considered to be 100% activity, while 0% complement activity was defined as the signal produced by 2% NHS treated with 50 μM FUT-175 inhibitor. Results were obtained from four parallel measurements. In all serum tests, the signal developing reaction on the plate was stopped when optical density of the highest signal intensity non-inhibited reaction was between OD 0.5–1.0. The typical background signal was around OD 0.05.
Polyclonal rabbit anti-human C3c antibody (A0062; DakoCytomation) was used as the primary antibody in 2000-fold dilution and HRP-conjugated anti-rabbit antibody (A1949, Sigma) was used as the secondary antibody in 40,000-fold dilution.
The signal produced by non-inhibited NHS was considered to be 100% activity, while 0% complement activity was defined as the signal produced by 2% NHS treated with 50 μM FUT-175 inhibitor. Results were obtained from four parallel measurements. In all serum tests, the signal developing reaction on the plate was stopped when optical density of the highest signal intensity non-inhibited reaction was between OD 0.5–1.0. The typical background signal was around OD 0.05.
10
Complement C3 Deposition in Rat Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four percent paraformaldehyde-fixed paraffin-embedded kidney sections (4 μm) were stained for complement C3 deposition in rat kidney. Rabbit anti-human complement C3c (1:500, A0062; Dako, Ely, Cambridge, UK), which cross-reacts with rat complement C3c, was followed by EnVision TM + Rabbit HRP (Dako, K4003, Ely, Cambridge, UK). Specifically bound antibodies were detected using a DAB Substrate kit (Vector, Burlingame, CA, USA). Sections were counterstained with Mayer's hematoxylin. Mounted sections were scanned and examined using Aperio ImageScope v11.2.0.780 software (Leica Biosystems Inc., Buffalo Grove, IL, USA).
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!