In situ hybridization of ERα mRNA in osteoclasts was performed on FFPE bone sections (n = 4 per group) from the lumbar spine using the RNAScope 2.5 HD Reagent kit (Advanced Cell Diagnostics [ACD], Newark, CA, USA). Then 5‐μm‐thick paraffin sections were deparaffinized, followed by Pepsin Reagent (Sigma) antigen retrieval for 30 min at 37°C. Target probes for ERα (Esr1) (Catalog No. 478201, ACD) and Oscar (Catalog No. 1179641‐C1, ACD) were used with the RNAScope procedure followed according to the manufacturer's instructions. Sections were mounted (VectaMount, Vector Laboratories, Burlingame, CA, USA) and visualized using a ×40 objective of the Nikon Eclipse TI microscope. Approximately 200 osteoclasts were counted per section (in 30 separate fields of view) and scored for ERα positivity.
Pepsin reagent
Manufactured by Merck Group
Sourced in France
Pepsin Reagent is a laboratory product manufactured by Merck Group. It is a proteolytic enzyme derived from porcine gastric mucosa that is commonly used in biochemical research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Vectamount, by Vector Laboratories (2 mentions)
Rnascope 2.5 hd reagent kit, by Advanced Cell Diagnostics (2 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Evos m5000 light microscope, by Thermo Fisher Scientific (1 mentions)
Elastin, by Merck Group (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Elastin, by Abcam (1 mentions)
Bloxall endogenous blocking solution, by Vector Laboratories (1 mentions)
Blockaid blocking solution, by Thermo Fisher Scientific (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
4 protocols using pepsin reagent
1
Quantifying Estrogen Receptor Alpha in Osteoclasts
2
Osteocyte ERα Expression via In Situ Hybridization
3
Immunohistochemical Evaluation of Type I Collagen Synthesis by DP-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Type I collagen synthesis by DP-MSCs was determined using immunohistochemistry as described previously 20 with slight modifications. Briefly, DP-MSCs were cultured for 7 days in fibrin-alone, fibrin/CLIN-PLA-NP or fibrin/PLA-NP hydrogels as described in Section 2.7. Hydrogels were fixed in acidified formal alcohol (AFA; Microm Microtech, France) at 7 1C for 24 h and classically embedded in paraffin. Five mm serial sections were cut, deparaffinized and rehydrated. For antigen retrieval, sections were incubated at 37 1C first in 0.5% hyaluronidase diluted in PBS for 1 h then in Pepsin Reagent (ref R2283, Sigma Aldrich, France) for 15 min. Endogenous peroxidase activity was blocked by incubating the sections in 0.5% hydrogen peroxide for 10 min at RT. To reduce non-specific binding of antibodies, sections were treated with BlockAid TM (Thermo Fisher Scientific, USA) at RT for 1 h. Sections were then incubated with the anti-type I collagen rabbit polyclonal antibody (1/2000) (ref. 20111[380k], Novotec, Bron, France) overnight at 4 1C. Antibody detection was performed with an ImmPRESS s HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection kit (Vector Laboratories, USA). Sections were finally counterstained with hematoxylin, mounted with DEPEX medium (ref 06522, Sigma Aldrich, France) and observed with an Eclipse TE300 microscope.
4
Immunohistochemical Detection of Elastin and CD3e
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After deparaffinization and rehydration, the antigen retrieval step was carried out by incubating 5 µm sections (i) in pepsin reagent (#R2283, Sigma–Aldrich, Saint Quentin-Fallavier, France) for 5 min at 37 °C for elastin immunodetection or (ii) in 10 mM sodium citrate buffer, pH 6, at 98 °C for 20 min for CD3e immunolabeling. Endogenous peroxidases were then quenched for 10 min at room temperature (RT) in Bloxall® Endogenous Blocking Solution (#SP-6000-100, Vector Laboratories, Burlingame, CA, USA), and non-specific sites were blocked for 30 min at RT with BlockAid™ Blocking Solution (#B10710, Invitrogen™, Illkirch-Graffenstaden, France). elastin (#21600 (1/2000), Abcam, Waltham, MA, USA) or CD3e (#MA1-90582 (1/200), ThermoFisher, Waltham, MA, USA) primary antibodies were incubated in a blocking solution overnight at 4 °C, and biotinylated secondary antibodies were incubated for 30 min at RT. Revelation was performed using ABC Reagent and 3,3′-Diaminobenzidine (DAB) chromogen (R.T.U. Vectastain Universal Elite ABC Kit from Vector Laboratories, #PK-7200, Burlingame, CA, USA), as recommended by the manufacturer, and nuclei were counterstained using hematoxylin (#H-3401-500, Vector Laboratories).
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!