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coverslip

Coverglass
German glass
german glass can be used for growing and culturing cells that normally hvae poor adhesion to plastic or regular glass surfaces.
borosilicate
quartz
for application in requisition of absolute UV transparency and high temperature properties. windows of transparenty from 150nm to 500nm. 

Marienfeld 24x60mm high precision No.1.5H coverslip
chemical resistant borosilicate glass D263 M 
thickness 0.170mm+/- 0.005mm



slides or coverslips used in fluorescence microscopy are required to be extremely clean.
newly opened coverslips may look clean but may have a thin film of grease that may interfere with processing steps and tissue culturng.
so the coverslips need to be cleansed with acid or base solutions.

Coating procedures such as gelatin, aminoalkylsilane or poly-L-lysine solution to promote adhesion of cells to glass surface.
gelatin or aminoalkylsilane is usually used for tissue sections or small organisms, whereas poly-L-lysine is routinely used for culturing cells.

CSHL protocols

some other links

Cleaning

The commercially purchased glass slides or coverslips may contain grease to ease the taking. However, this grease could interfer with experimental process.
It is imperative that the slides and the coverslips used in fluorescence microscopy needs to be cleaned.

glass cleaning can be achieved by acidic, basic solutions, or detergents.

Acids:
  1. 300mL of HNO3:HCl =2:1 (solution is orange-red) !! be sure to operate in fume hood.
  2. place coverslips in the acidic solution for 2hr with occasional swirling.
  3. decant the acid carefully in inorganic acid waste receptacle.
  4. wash the coverslips with ddH2O
  5. store in 70% ethanol
alternate protocols in our lab
  1. 400mL of H2SO4:H2O2=3:1 in fume hood
  2. immerse the coverslips or slides in the solution for 15minutes
  3. type I water wash for 3 times.
  4. coverslips in type I water sonicate for 15minutes
  5. type I water wash for 3 times.
  6. (nitrogen air blow dry.)
  7. store in 70% ethanol.
  • !! the oxygen from the hydrogen peroxide would continuously produced for a week. keep the waste in fume hood until no bubbles forming. then decant the waste in inorganic acid waste receptacle.
  • To confirm good cleaning, when you take one coverslip out from the water, you can see a thin water film on the coverslip.
Basic:
  1. incubate coverslips in 2N NaOH for 2hrs.
  2. decant the basic in basic waste receptacle
  3. rinse extensively in H2O
  4. store in 70% ethanol.
Detergents: (TFD4, Franklab, France)
  1. 1:20 dilution of TFD4 detergents in ddH2O
  2. 20 minutes wash in sonication washer.
  3. Type I water 20minutes wash in sonication washer
  4. repeat step 3 3 times.
RCA clean


Poly-L-Lysine Coating Protocols

Poly-L-Lysine 
  1. 500µg/mL Poly-L-Lysine in Type I water (For some types of cells, higher concentration may be required (1mg/mL)
  2. coat slides by dipping into poly-L-lysine solution or apply enough to cover glass solution
  3. incubate 10min at room temp.
  4. wash 3x with sterile H2O, allow air dry.
  5. UV sterilization for 1hr
poly-L-lysine coated coverslips can be made more hospitable to cell attachment by the addition of a coating of laminin (2-5mg/cm2) air dried coated coverslips at least 45 min prior to plating. it is not advisable to store laminin coated coverslips

0.5mL for 25cm^2

other protocols:


Gelatin Subbing Protocols

Gelatin

Preparation of subbing solution:
  1. dissolve gelatin in type I water in a flask or beaker at 60℃ to make a 0.2% (w/v) solution.
  2. cool the solution to 40℃, add chromium potassium sulfate to 0.02% (w/v)
  3. cool the solution to 4℃, use immediately or store in 4℃ for a few weeks.
subbing protocols:
  1. place cleaned slides or coverslips in appropriate racks and immerse in subbing solution for 2 minutes at 4℃
  2. avoid bubbles on the surfaces.
  3. remove the slides and dry slides overnight in laminar flow.
  4. the subbed slides and coverslips would be stable at room temp for a few weeks

Collagen Coating Protocols

Collagen

Thermo Fisher A1048301 rat tail collagen 20mL
3mg/ml

I. coating protocol from Thermofischer
Gelling procedures
a. place collagen (3mg/ml), sterile 10X PBS or 10X medium 199, sterile distilled water and sterile 1N NaOH on ice
b. determine the concentration and final volume of collagen needed for experiment
c. determine the amount of reagents needed so that collagen is at the desired concentration in 1X PBS or M199 wiht normal osmality and neutral pH
d. in a sterile tube mix the ddH2O, 1N NaOH and 10x PBS
e. slowly pipet the collagen into the tube, and gently pipet solution up and down to mix well. The resulting mixture should achieve a pH of 6.5-7.5 (optimum pH 7.0)
f. dispense the collagen into the desired plates or dishes immediately and store them on ice. Gelling may occur rapidly at room temperature.
g. incubate at 37°C in humidifed incubator for 30-40 minutes or until a firm gel is formed
h. rinse the gel with sterile 1X PBS or cell culture medium before seeding cells


II. thin coating procedure
Optimization for desired protein concentration may be required. Astarting concentration of 5µg/cm2 is recommended.
a. determin the volume needed for experimentation
b. dilute the collagen to 50µg/mL in 20mM acetic acid at the final volume needed
c. add solution to plates or dishes at 5µg/cm2 
d. incubate at room temperature for 1 hour
e. carefully aspirate solution from the well or dish
f. rinse dish three times with equal volume of sterile PBS or media to remove the acid
g. plates may be used immediately or air dried and stored at 2-8°C for future use.


III. collagen coating of glass bottom dishes and glass bottom plates from Cell vis
http://www.cellvis.com/collagen_coating_of_glass_bottom_dish_plate.php
dilute the stock solution to 50µg/mL with 0.02M acetic acid or 0.01M HCl
add working solution into glass bottom at 5µg/cm2. 
incubate for 1hr at RT in BSC
aspirate collagen solution
rinse 3 times with PBS or serum free media
air dried and kept at 4°C for a month

IV. collagen coating procedure from NeuVitro

V. Collagen gelling procedure from GRACE biolabs



Silanizing protocols:



Polystyrene slides:
tissue culture treated polystyrene slides can be used to grow adherent cells.

Thermanox 
Thermanox are made from members in polyolefin family. It is highly resistant to most chemicals, alcohols, aldehydes, hydrocarbons, dilute acids (<10%) and dilute alkalis (<2%).
limited resistance to chlorinated hydro-carbons. Not resistant to concentrated acids or bases.
culture treated on one side for enhanced cell attachment and growth.
auto-fluorescent in the range 380 to 545nm.
autoclavable 20mines steam, 20minutes dry at 120℃

Rinzl
optical clear vinyl with refractive index of glass.
ideal for wet mount and urines.
not autofluorescent.

Permanox slides
permanox is a strong, biologically inert material resistant to non-chlorinated hydrocarbons, acetone and other EM reagents.
permanox slides allow cell fixation and cell staining on the slide. 
non-autofluorescent.



Coverslip for use in confocal microscopy.
The thickness of the coverslip used in fluorescence microscopy is crucial.
Most objectives are designed to be compatible with No.1.5 glass coverslips.

a resources in nikon microscopy resource shows how crucial matching the thickness is imperative especially when using high numerical aperature objectives.
some special objectives are designed with a correction collar to correct this.


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