The largest database of trusted experimental protocols

5 protocols using 3 amino 9 ethylcarbazole aec substrate

1

Quantifying Intestinal IgA-Secreting Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
The frequencies of intestinal IgA+ ASCs and VP6+ IgA+ ASCs were determined by ELISPOT as described (20 (link)). Briefly, 96-well plates (Millipore) were coated with affinity-purified goat anti-human IgA + IgG + IgM (H + L) (KPL) at a concentration of 4 μg/ml. Wells were coated with phosphate-buffered saline (PBS) as a negative control. Plates were incubated overnight at 4°C and blocked for 2 hours at 37°C with complete medium before use. B cells were suspended in complete medium containing peroxidase-conjugated goat anti-human IgA (Sigma-Aldrich), distributed in ELISPOT plates, and incubated for 4 hours at 37°C in 5% CO2. Plates were washed and developed with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories). The total ASCs per well was determined by counting the spots using the ImmunoSpot Analyzer (Cellular Technology Limited). Background ASCs detected in the wells coated with PBS were subtracted from the quantities of ASCs in treated wells.
+ Open protocol
+ Expand
2

Immunocytochemistry of TLR7 in 3T3-L1 Adipocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
The 3T3-L1 fibroblasts were differentiated into mature adipocytes, as described above, and shock-frozen by ice-cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. The air-dried cells were incubated in PBS for rehydration. The endogenous peroxidase activity was blocked with 3% H2O2 (ROTH, Karlsruhe, Germany). To avoid non-specific protein binding, the cells were incubated in 10% bovine serum albumin (BSA, ROTH, Karlsruhe, Germany), 10% fetal calf serum (FCS, Sigma-Aldrich, Steinheim, Germany) and 10% chicken serum (Sigma-Aldrich, Steinheim, Germany), followed by 3 h incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad, CA, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.
+ Open protocol
+ Expand
3

Immunohistochemical Analysis of TLR7 in Adipose Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
The murine and human adipose tissue samples were fixed in ROTI®Histofix 4% (Carl Roth, Karlsruhe, Germany) for 24 h and later embedded in paraffin. The paraffin was removed with xylene (ROTH, Karlsruhe, Germany) and by consecutive washing steps with 100%, 96% and 70% ethanol (ROTH, Karlsruhe, Germany). The endogenous peroxidase activity was blocked with 3% H2O2. Tissue samples were incubated for 60 min in citrate buffer at 60 °C. Non-specific binding sites were blocked with 5% BSA for 60 min in a humid chamber followed by an overnight incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad California, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, California) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.
+ Open protocol
+ Expand
4

Histological Analysis of Nasal Turbinates

Check if the same lab product or an alternative is used in the 5 most similar protocols
The nasal cavities from each animal were taken to perform histologic studies on turbinate tissue sections. Under gentle agitation, samples were first fixed in 4% paraformaldehyde for 3 days at RT, then decalcified at 4°C in 10% EDTA (Sigma) at pH 7.4 for 7 days, and cryopreserved at 4°C in sucrose 30% (Sigma) for 3 days. After immersion in optimum cutting temperature compound (OCT, TissueTek, Sakura Finetek), the samples were quickly frozen and stored at -80°C until processing. Sagittal sections, 8-μm thick, were performed using a cryostat microtome (CM1860, Leica). Sections were successively incubated at RT with acetone, 3% hydrogen peroxide (Sigma), PBS containing 3% bovine serum albumin and 5% normal goat serum, and for 1 hour with HRP-conjugated anti-FIX antibody (1:100, goat polyclonal, Affinity Biologicals). HRP was revealed using 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories). Sections were counterstained with Hematoxylin QS (Vector Laboratories), mounted with Glycergel (Agilent Technologies). Images of nasal sections were captured using an inverted microscope (40× oil lens and 10-μm scale bar, Nikon Ti-E microscope).
+ Open protocol
+ Expand
5

Immunocytochemical Analysis of TLR9 in Adipocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
3T3-L1 fibroblasts were differentiated into mature adipocytes as described previously. The shock-frozen sections were fixed with ice cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. Air-dried cells were incubated in PBS for rehydration. Endogenous peroxidase activity was blocked with 3% H 2 O 2 (ROTH). To avoid non-specific protein binding, cells were incubated in 10% bovine serum albumin (BSA, ROTH), 10% fetal calf serum (FCS, Sigma-Aldrich) and 10% chicken serum (Sigma-Aldrich), followed by 3-h incubation in a moist chamber with rabbit anti-mouse TLR9 antibody (5 µg/mL in 1% BSA; ab37154; Abcam). Cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (2.5 µg/mL in 1% BSA; P0448; DAKO) for 90 min. Color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. Rabbit isotype-matched IgG sera (ab27478; Abcam) served as an isotype control. Parallel experiments without primary antibody were carried out as negative controls.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!