Archive for November, 2008

Immunohistochemical staining protocol

November 28, 2008
Tagny Duff

Photo:Tagny Duff

Here is the protocol used for the immunohistochemical staining process applied in the Living Viral Tattoos project. It will be adapted for the Cryobook Archives. The protocol was developed by Dr. Stuart Hodgetts.

I think about this scientific protocol as a recipe or a kind of text score. While I did follow the protocol in order to learn the technique of immuno staining– for the most part I deviated from it slightly. It is very similar a process to cooking, printing and painting. Protocols, such as this one, can be hacked, altered, repurposed for artistic means and critical reflective engagement with the tools of biotechnology.

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Viral Tattoos Recipe
For visibly blue expression of transfected cells on epidermis

Determine Multiplicity of infection (MOI) based on amount of viral vector to cell culture ratio. We used 1.0x 10 4 Transducing particles (TU) per ml and then divided accordingly to test for best MOI rate.

1. Suspend cells grown in flasks with 5 ml Trypsin.
2. Add DMEM medium with 10% FBS
3. Count cells
4. Add 1x 10 (power of 4) cells to 1 ml cell culture in each 24 well dish. (Optional – add Poly-L-Lysine for stronger adhesion of cells on plates).
5. Put dish in incubator overnight (12 hours)
6. Check that cells are semi-confluent (60 % is best).
7. Put fresh medium (1 ml) in the dishes. Put 2ml of medium in first dish (Nat)
8. Take 10 microliters of lentivirus and put in first dish.
9. Pipette 1ml into second dish—1ml into the third to the sixth well (to dilute the viral nate. (1/2, 1/4, 1/8, 1/16, 1/33)
10. Put 1.1 ml in the second dish. Take another 10 microliters of lentivirus. Take 100 microliters into each of the other six dishes. ( 1/10, 1/100, 1/000, etc)
11. Add polybrane ( 8 ug/ml)
12. Seal dish with parafilm and put in incubator for 4 to 12 hours.
13. Replace medium (without FBS)
14. Place in incubator for 12 hours.

Option: Skip the cell count and hope for the best. Add as much or as little quantity of virus as you feel like to the cell culture.

Prepare skin- Wash in PBS. Cut sections with sterilized scissors prior to suspending viral cells.

15. Remove medium from dishes
16. Suspend virus and cells with trypsin (fill bottom of dish) for approx 2 mins or until you see cells lifting.
17. Add medium

Place skin samples into dishes. Pipette cells onto the surface. (Separately each skin sample with one cell dish to determine MOI with microscopy, if desired).Try to keep the concentration of cells in one area for dense expression of immunostains. (Ie. Bluer colour concentration).

Immunostaining pig and human skin (Stuart Hodgetts)

1. “Seed” skin with cells and virus in about 1ml of medium – leave for at least 4 hrs
2. Fix tissue with 1:1 acetone:methanol (volume:volume or v/v) for 5-10 mins
3. Incubate with primary antibodies for 30 mins at 37°C
4. Rinse 3x with phosphate buffered saline (PBS) or medium (serum free)
5. Incubate with secondary antibodies for 30 mins at 37°C
6. Rinse 3x with L-15
7. Rinse 3x with PBS or medium (serum free)
8. Develop with 4-chloro-1-naphthol (4CN) chromagen solution until desired staining is achieved
9. If required, rinse and leave submerged in tap water to intensify 4CN staining

Primary antibodies:
Rabbit anti-mouse desmin at 1/400 dilution in PBS
Mouse anti-mouse vimentin (monoclonal autoantibody (clone C10) obtained from Stuart Hodgetts, Raised in NZB/W F1 autoimmune mice) – used undiluted

Secondary Antibodies:
Goat anti-Rabbit Ig – horse radish peroxidase conjugated at 1/2000 in PBS
Donkey anti-mouse Ig – horse radish peroxidase conjugated at 1/2000 in PBS

4CN Chromagen:
20mg 4CN dissolved in 20ml methanol = Solution A
50mM NaCl/Tris-HCl solution = Solution B
Mix 1:5 solutions A:B then add 0.1ml hydrogen peroxide, mix again
Use 50ml

Additional notes on skin:
Pig skin is tough and the cells need to be pipetted in one area in order to render colour. The cells expresses more easily in the fibreblasts found in the dermal tissue of both human and pig skin.
It is recommended to submerge both types of skin in tap water for at least two days for more intensity of staining.

Plans for making the cryobook archives

November 28, 2008

Tagny Duff

Stage 1: The making of the Cryobooks
( SymbioticA, the art and science collaborative research laboratory, Perth, Western Australia).

-I will meet with plastic surgery patients to ask for consent to use post-surgery tissue waste. I have established a working relationship with a plastic surgeon and will submit the necessary human ethics and biosafety approval forms to SymbioticA and the University of Western Australia required to acquire post-surgery tissue for artistic research purposes. (All approval forms were obtained for the previous Living Viral Tattoos project).

-New immunohistochemical staining techniques will be further tested in vitro and developed to express a range of colour stains on skin. (This will extend research previously conducted with immunohistochemical staining processes as seen in the video “Moist Media Archives: Living Viral Tattoos”.

-I will experiment with cutting the shape and size of skin in order to make the pages. I will work a cryostat machine which will allow me to cut tissue.

-I will also take a workshop to learn techniques for sewing sutures in skin in order to bind the cryobooks together. Different techniques for fixing and preserving the books will be tested.

– A series of cryobooks will be made based on the aforementioned techniques within a time frame of one month.

Stage 2: The making of the “Cryobook archive” prototype
A cryogenic display case for the cryobooks will be designed and developed, as one of the two main components of the installation. The Cryobook Archives will be designed and engineered to facilitate the public interaction with the cryobooks.

Phase three: The testing of prototypes in situ

Testing of the functionality and aesthetic display of the Cryobook Archives prototype and the cryobooks will occur in a designated laboratory space. The laboratory space will be explored and researched as an in situ performance/exhibition site. Once all the safety requirements, artistic and technical elements are complete, visitors will be invited to engage with the prototypes for a one-day public engagement.

This last phase will provide research into the range and scope of interactivity enacted between audiences, the in situ context and the installation. It will also provide a “safe space” to test safety measures for audiences and realize the technological functionality of the installation components.

Documentation of previous research-creation

November 28, 2008
Tagny Duff

Photo: Tagny Duff

This breast tissue was donated from a patient undergoing elective breast surgery reduction to the Living Viral Tattoo Project. Immunohistochemical stains were used to render areas of viral transfection on the surface of the skin. ( Also see the link to my video “Living Viral Tattoos” on my website. The video is currently being screened as part of Evolution Haute Couture, curated by Dmitry Bulatov).

Tagny Duff

Photo: Tagny Duff

Tagny Duff

Photo:Maria Grade Godinho