Following de-paraffinization and rehydration, sections were incubated with 3% (v/v) H2O2 for 5 min at room temperature to quench endogenous peroxidase activity. Sections were then blocked with 1% BSA for 5 min at 4 °C and incubated with the relevant primary antibody diluted in 1% BSA in PBS overnight at 4 °C. Sections were then washed three times in 0.5% Tween-20 and incubated for an hour with the relevant biotinylated secondary antibody and ExtrAvidin–peroxidase (Sigma, E2886 1:50). Visualisation of the immune complex involved incubation with 3-amino-9-ethylcarbazole (AEC), resulting in a reddish-brown reaction product. Sections were counter stained with Alcian Blue 8GX except when the antibody against Aggrecan was used. For the SOX9 (Millipore, AB5535 1:150,) and Aggrecan (R&D, AF1220 1:240) antibodies, antigen retrieval was performed using 10 mM citrate buffer at 95 °C for 30 min. For immunostaining using anti-Type 1 collagen antibody (gift from Dr Larry Fisher, 1:1000) and the anti-Type II collagen (Calbiochem, 2341871:500) antibody, sections were treated with Hyaluronidase (0.8 mg/ml) at 37 °C for 20 min in order to unmask epitopes rendering them accessible for immunostaining. Microscopy was performed using a Zeiss Axiovert microscope and Axiovision imaging software (Carl Zeiss, Cambridge UK).
E2886
Manufactured by Merck Group
Sourced in Germany
E2886 is a laboratory equipment product offered by Merck Group. It serves as a precision weighing instrument used for accurate measurement of small masses or weights. The core function of E2886 is to provide reliable and consistent weight determination for various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Extravidin peroxidase, by Merck Group (2 mentions)
Ab5535, by R&D Systems (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
Axiovision imaging software, by Zeiss (1 mentions)
Ab188453, by Abcam (1 mentions)
Ab188470, by Abcam (1 mentions)
Ab191698, by Abcam (1 mentions)
Ab124297, by Abcam (1 mentions)
Ab194296, by Abcam (1 mentions)
Ab109411, by Abcam (1 mentions)
Biotinylated goat anti rabbit igg antibodies, by Jackson ImmunoResearch (1 mentions)
Sk 4105, by Vector Laboratories (1 mentions)
B7264, by Merck Group (1 mentions)
Mab318, by Merck Group (1 mentions)
Pr1210, by Solarbio (1 mentions)
Dpx mountant, by Merck Group (1 mentions)
Extravidin, by Merck Group (1 mentions)
Mz6 microscope, by Leica (1 mentions)
Dmrb light microscope, by Leica (1 mentions)
D5637, by Merck Group (1 mentions)
7 protocols using e2886
1
Immunohistochemical Staining Protocol
2
Protein Extraction and Immunoblotting
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Isolation of total protein from the frozen tissues and denaturing electrophoresis were performed according to the Lamely method using the Bio-Rad system (USA). The protein concentration was measured in each sample, and equal amounts of protein were applied to each well. Transfer to the nitrocellulose membrane was carried out according to Towbin et al.34 (link),35 (link).
To evaluate each protein, specific antibodies based on rabbit immunoglobulins were used at the following concentrations recommended by the manufacturer: 1:1000 for S-phase methylation of Dnmt1 (Abcam, #ab188453); 1:2000 for de novo Dnmt3a methylase (Abcam, #ab188470); 2 μg/ml for T-methylcytosine hydroxylase (demethylase) TET1 (Abcam, #ab191698); 1:1000 for TET2 (Abcam, #ab124297); 1:1000 for histone acetylase KAT1/HAT1 (Abcam, #ab194296); and 1:1000 for HDAC1 deacetylase (Abcam, #ab109411). Biotinylated goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Lab., Inc., #111-035-003) were used as secondary antibodies at a dilution of 1:10,000. The membranes were then treated with streptavidin solution conjugated with horseradish peroxidase (Sigma, #E2886) at a dilution of 1:10,000. The protein bands were revealed using 3,3′-diaminobenzidine (Amresco, #E733-50), and the data were analyzed in the ImageJ program.
To evaluate each protein, specific antibodies based on rabbit immunoglobulins were used at the following concentrations recommended by the manufacturer: 1:1000 for S-phase methylation of Dnmt1 (Abcam, #ab188453); 1:2000 for de novo Dnmt3a methylase (Abcam, #ab188470); 2 μg/ml for T-methylcytosine hydroxylase (demethylase) TET1 (Abcam, #ab191698); 1:1000 for TET2 (Abcam, #ab124297); 1:1000 for histone acetylase KAT1/HAT1 (Abcam, #ab194296); and 1:1000 for HDAC1 deacetylase (Abcam, #ab109411). Biotinylated goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Lab., Inc., #111-035-003) were used as secondary antibodies at a dilution of 1:10,000. The membranes were then treated with streptavidin solution conjugated with horseradish peroxidase (Sigma, #E2886) at a dilution of 1:10,000. The protein bands were revealed using 3,3′-diaminobenzidine (Amresco, #E733-50), and the data were analyzed in the ImageJ program.
3
Unbiased Stereological Analysis of Tyrosine Hydroxylase-Positive Neurons
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For TH immunohistochemistry, primary and secondary antibody solutions as well as ExtrAvidin solutions were prepared in phosphate buffer (PB) and 0.3% Triton X-100. Each incubation step was followed by three 5-min rinses in PB. Sections were incubated 48 hours at 4°C in a solution with a mouse anti-TH antibody (1:1000; MAB318, Merck Millipore) and then incubated for 2 hours with a biotinylated goat anti-mouse antibody (1:200; B-7264, Sigma-Aldrich). Furthermore, sections were incubated for 1 hour in an ExtrAvidin solution (1:500; E2886, Sigma-Aldrich). As chromogen, peroxidase was used (SK-4105, Vector). Last, sections were mounted on chromalin-coated slides, air-dried, dehydrated, and coverslipped. Four animals per group were used for stereological analysis that was performed on brain sections containing the SNpc. Using the Stereo Investigator System (MicroBrightField Europe e.K., Magdeburg, Germany), a three-dimensional optical fractionator counting probe (x, y, and z dimensions of 50 μm by 50 μm by 20 μm, respectively) was applied to obtain unbiased estimates of total TH+ cells according to published procedures (37 (link)). Data collecting for stereology was done by the experimenter blind to the group analyzed.
4
Quantification of Allergen-IgE Complexes
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In brief, 20 μl of indicator serum exhibiting high IgE concentrations for Artemisia (> 100 kU/l) was incubated with 20 μl of sample serum in the presence of Artemisia pollen extracts (XP61D3A2.5, Stallergenes Greer, USA) for 1 h at 37 °C, allowing allergen-IgE complex formation. Adding 20 μl of RPMI 1640 medium (SH30809.01, Hyclone, USA) instead of sample serum served as control. For complex formation the optimal antibody/allergen ratio was found at concentrations of 1:500 by applying the appropriate indicator serum (see detail in Additional file 1 ). Allergen-IgE complexes were transferred to microtiter plates coated with soluble CD23 protein (123-FE-050, R&D systems, USA) and incubated for 1 h at room temperature. After addition of biotin-conjugated anti-human IgE antibody (555858, BD Biosciences, Germany), streptavidin-peroxidase (E2886, Sigma-Aldrich, Germany), and 3,3′,5,5′-tetramethylbenzidine (PR1210, Solarbio, China), allergen-IgE complexes bound to immobilized CD23 were determined at 450 nm using the microplate reader. All samples were measured in duplicate. Data were expressed as binding of allergen-IgE complexes relative to the binding with indicator serum alone, calculated as:
5
Immunohistochemical Staining of mGluR1a
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DAB-peroxidase IHC was performed as described by Kwakowsky et al. (2018) (link). In brief, sections were washed in PBS with 0.2 % Triton X-100 (PBST) before blocking for endogenous peroxidases (50 % methanol and 1 % H2O2) for 20 min, followed by three 10-minute washes in PBST and incubated for 72 h in mGluR1a primary antibody in immunobuffer at 4 °C (Table 3 ). The sections were then washed in PBST before incubation for 24 h with the biotinylated secondary antibody (anti-sheep IgG-Biotin antibody produced in donkey, 1:1000, B7390, Sigma, St. Louis, MO, USA) in immunobuffer at room temperature (RT). The sections were then washed in PBST before incubation with ExtrAvidin (1:1000, E2886; Sigma, St. Louis, MO, USA) in immunobuffer for 4 h at RT, followed by three 10-minute washes in PBST before development in 0.05 % DAB and 0.01 % H2O2 in 0.1 M phosphate buffer. Sections were washed in PBST and mounted onto glass slides, dried, dehydrated through a graded series of ethanol, and cleared in xylene. The slides were coverslipped with DPX mountant (1019790500; Merck, Whitehouse Station, NJ, USA). The sections were imaged on either a Leica DMRB light microscope or a Leica MZ6 microscope (Wetzlar, Germany).
6
Immunohistochemistry of GFAP and CLN Proteins
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Sections were stained with rabbit anti-cow glial fibrillary acidic protein (GFAP; 1:5000, Z0334 in 50% methanol in PBS (GSB4), 30 min, room temperature. Sections were then pre-incubated in 15% NGS in PBST prior to overnight incubation at 4˚C in primary antibody/lectin. Immunoreactivity was detected using biotinylated goat anti-rabbit IgG (1:1000; B7389; Sigma-Aldrich), 2 h, room temperature, followed by ExtrAvidin peroxidase (1:1000; E2886; Sigma-Aldrich) for 2 h at room temperature. Staining was visualized by incubation in 0.05% 3, 3′-diaminobenzidine (DAB; D5637; Sigma-Aldrich) and 0.01% H 2 O 2 in PBS, 7 min (GFAP, CLN5, CLN6) or 5 min (GSB4). Negative control sections, in which either the primary or secondary antibody was omitted, were included in all staining runs. Sections were mounted in chrome alum solution, air-dried, dehydrated in 100% ethanol, cleared in xylene, and coverslipped with DPX. Further unstained sections were mounted as above, air-dried, and coverslipped with glycerol for fluorescent microscopy.
7
Western Blot Analysis of HLA-G
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Total cell lysates were prepared using the Total Protein Isolation kit (Fisher Scientific GmbH, Germany). Protein concentrations were assessed by Bradford. For the analysis of HLA-G expression, 40 mg total proteins were loaded onto 12% polyacrylamide gels and resolved by SDS-PAGE under reducing conditions (5% b-mercaptoethanol). Separated proteins were then electro-transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia, Buckinghamshire, UK), followed by blocking of nonspecific binding sites using 5% nonfat milk in PBS for 1 h at RT. Blots were incubated with biotinylated anti-HLA-G antibody (Ab) (MEM/G1 Exbio, Germany; 1:1500) o.n. at 4 C, which was then detected with an HRP-streptavidin conjugate (E2886, Sigma Aldrich, Germany; 1:3000) for 1 h at RT. Reactions were developed using the enhanced chemiluminescence (ECL) detection reagent on a Bioimaging analyzer (LAS-1000; Fujifilm, Tokyo, Japan). Equal loading was confirmed by blotting the same membranes with an HRP-conjugated anti-b actin Ab (Sigma Aldrich, Germany; 1:50000). Densitometric analysis of the protein bands was performed using the Gel Analyzer Tool of the Image J software (National Institutes of Health, http://rsb.info.nih.gov/ij/), and relative HLA-G expression was calculated by normalizing to b actin expression as loading control.
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