The largest database of trusted experimental protocols

4 protocols using streptomyces hyaluronidase

1

Lubrication Testing Materials Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Reagents for lubrication testing materials were as described previously,2 (link),6 (link),33 (link) including HA in a 4000-kDa form (Healon; Advanced Medical Optics). The antibody to PRG4 was anti-Lubricin from AbCam,2 (link) nonspecific rabbit IgG was from Pierce, and mouse anti-rabbit IgG secondary antibody was from Jackson ImmunoResearch. Streptomyceshyaluronidase was from Seikagaku, SeaKem gold agarose was from Lonza; 50X TAE (2M Tris, 0.5 M ethylenediamine tetraacetic acid [EDTA]) electrophoresis buffer was from Life Technologies, Hybond-P polyvinylidene difluoride (PVDF) membrane for Western blotting was from GE Healthcare, and Stains-All was from Sigma-Aldrich.
+ Open protocol
+ Expand
2

Reagents for Lubrication Testing

Check if the same lab product or an alternative is used in the 5 most similar protocols
Reagents for lubrication testing materials were as described previously,2 (link),6 (link),33 (link) including hyaluronan (HA) in a 4,000 kDa form (Healon®, Advanced Medical Optics, Santa Ana, CA). The antibody to PRG4 was anti-Lubricin from AbCam (Cambridge, MA);2 (link) nonspecific rabbit IgG was from Pierce (Rockford, IL); mouse anti-rabbit IgG secondary antibody was from Jackson ImmunoResearch (West Grove, PA). Streptomyces hyaluronidase was from Seikagaku (Tokyo, Japan). SeaKem® gold agarose was from Lonza (Rockland, ME); 50X TAE (2M Tris, 0.5M EDTA) electrophoresis buffer was from Life Technologies (Carlsbad, CA); Hybond™-P polyvinylidene difluoride (PVDF) membrane for Western blotting was from GE Healthcare (Piscataway, NJ). Stains-All was from Sigma-Aldrich (St. Louis, MO).
+ Open protocol
+ Expand
3

Immunofluorescence Staining for LANA and HA

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were incubated in 1:1 methanol-acetone at 20°C for fixation and permeabilization, followed by a blocking reagent (10% normal goat serum, 3% bovine serum albumin, and 1% glycine) for an additional 30 min. Cells were then incubated for 1 h at 25°C with 1:1000 dilution of a rat anti-LANA monoclonal antibody (ABI, for LANA wt) or a mouse anti-V5-Tag monoclonal antibody (Cell Signaling, for LANA deletion fragments) followed by 1:100 dilution of a goat anti-rat or goat anti-mouse secondary antibody conjugated to Texas Red (Invitrogen). For intracellular HA detection, cells were permeabilized for 20 min at room temperature with 0.1% Triton-X-100 in 1% BSA, and incubated overnight at 4 °C with bHABC (biotinylated hyaluronan binding complex, Sigma) (1.25μg/mL) in 1% BSA. To remove the pericellular HA, fixed cells were treated with Streptomyces hyaluronidase (1 turbidity reducing unit/mL, Seikagaku Kogyo) before permeabilization. After washing, the cells were incubated for 1 h with Alexa 488-labeled streptavidin (1:1000) (Invitrogen) for bHABC staining. Cells were counterstained with 0.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI, Sigma) in 180 mM Tris-HCl (pH 7.5) for nuclear localization. Slides were washed once in 180 mM Tris-HCl for 10 min and prepared for visualization using a Leica TCPS SP5 AOBS confocal microscope.
+ Open protocol
+ Expand
4

Immunohistochemical Detection of Hyaluronan

Check if the same lab product or an alternative is used in the 5 most similar protocols
The tissue sections (5 µm) were first kept at 58 °C for 30 min, after which deparaffinization was accomplished by incubating in 1% H2O2 for 5 min to block endogenous peroxidase. Sections were washed with water and phosphate buffer (PB). Non-specific binding of the antigen was blocked by incubating in 1% bovine serum albumin (BSA) in PB at 37 °C for 30 min. The sections were incubated overnight at 4 °C in 1:30 dilution of 50 µL/mL biotinylated hyaluronan binding complex (bHABR), prepared as described by Tammi et al. [19 (link)]. The sections were washed with PB, and the bound HA binding complex was visualized with the avidin-biotin-peroxidase (ABC) method (1:200, Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were subsequently washed with PB. Diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) was used as chromogen, containing 0.5 mg/mL PB and 0.03% H2O2, incubating for 5 min. The sections were washed with distilled water and counterstained with Mayer’s hematoxylin for 1 min. The sections were washed, dehydrated, and mounted in DPX (BDH Laboratory Supplies, Poole, UK). To control the specificity of the staining, the specimens were predigested with Streptomyces hyaluronidase (100 TRU/mL in acetate buffer, pH 5.0 for 3 h at 37 °C; Seikagaku, Kogyo, Tokyo, Japan) in the presence of protease inhibitor.
+ 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!