I am working with human meniscus cells . In high density cell seeding with biomolecules that promotes growth I got a thin membrane as given in image and H&E staining of the same. Is it a tissue formation? Can anybody help me.
Difficult to say what it is. If you want to know, you will have to look at it histologically. Put all or parts of it into a paraffin block and get it sectioned and stained.
For meniscus you need to have production of extracellular matrix which you can assess in histology for instance. If you have access to a second harmonic microscope you could also do some imaging of collagen fibrils if any is produces prior to sectioning for histology, which would make really nice pictures.
I would suggest you use Alcan blue at pH 1.0 followed by Saffranin-O at pH 2.5. As you know Alcan blue is blue, whereas Saffranin-O is an orange color, in combination they are a brown color. Therefore, when only sulfated proteoglycans are present (i.e., heparan sulfate, keratin sulfate) the tissue will be blue. When only carboxylated proteoglycans are present (i.e., non-sulfate chondroitin ) and hyaluronic acid the tissue will be orange. However, if you have both sulfate and carboxylated proteoglycans present (i.e., chondroitin sulfate, dermatan sulfate, and/or aggrecan ( which is a combination of chondroitin sulfate and keratan sulfate GAG chains), then the tissue is brown in color. Try staining articular cartilage. You will see zonation patterns of staining. The transitional, tangential, radial, tide water, and calcified cartilage zones all have different patterns of staining. Meniscus cartilage has at least two zones based on this carbohydrate histochemical staining, an inner zone and an outer zone. You can verify which proteoglycans you have by using PG-specific enzymes, i.e., heparanase for heparan sulfate, keratanase for keratan sulfate, hyaluronidase for hyaluronic acid, chondroitinase-AC for chondroitin sulfate and chondroitinase-ABC for chondroitin sulfate and dermatan sulfate. You can also distiguish meniscal cartilage by it's particular collagen composition. Meniscus is classified as fibrocartilage and as such contains type-I and type-XII collagens, similar to tendons and ligaments. Whereas, articular cartilage, similar to hyaline cartilage and growth plate cartilage contain type-II and type-IX collagens. You can obtain the antibodies from the Developmental Studies Hybridoma Bank at the University of Iowa.