Stock Reagents:        
0.1% Triton X-100        
50% Bleach            
Heptane (Light Sensitive)
Methanol (Light Sensitive)    
PBS (1X and 5X Solutions)    
1X PBS + 0.1% Triton

Solution Recipes:
Fix Solution (3.7% Formaldehyde):        NGS Block Solution (1% NGS with 1% NaN3):
75 uL of 37% Formaldehyde                 5 mL PBS with 0.1% Triton-X
150 uL of 5x PBS                               50 uL NGS (Normal Goat Serum) (See Freezer)
525 uL of ddH2O                                50 uL NaN3 (Sodium Azide) (See Fridge)
                                                       -Store in the 4°C Fridge; Keep on Ice when used

1° Antibody Solution:                        2° Antibody Solution:
350-X uL Block Solution                    400-X uL Block Solution
X uL of Primary Antibody                    X uL of Secondary Antibody
                                                    -Keep dark and on ice as much as possible

-Note: Some antibody stocks are in Glycerol, so mixing may be required
-When preparing Antibody Mixtures, spin the solution down for 2 minutes at 14000 rpm at 4°C.
• Spinning the Antibody solution reduces the non-specific binding of the Antibodies. The serum proteins (Igs) within the NGS bind the non-specific sites in the embryo. The antibodies can also aggregate, so spinning down will remove some of the aggregates.

(1) Collect embryos from an embryo collection plate ~4 hours after cup-flipping. Use a paintbrush moistened with 0.1% Triton X-100 to transfer the embryos into an Eppendorf tube containing 0.1% Triton X-100.
•The Triton X-100 is a non-ionic detergent which removes any yeast that transferred with the embryos.

(2) Wash embryos 2-3 times with 0.1% Triton X-100.
•The Triton X-100 is a non-ionic detergent which removes any yeast that transferred with the embryos.

(3) Remove wash, and add 50% Bleach to the cleaned embryos, and set them on the nutator for 4 minutes, then allow them to settle for 1 minute.
•The 50% Bleach solution effectively strips the Chorion layer of the embryos.
→ As a result, the embryos will appear smaller due to the removal of the Chorion and appendages.
•Note: If exposed to 50% Bleach for more than 5 minutes risks the degradation of layers beyond the chorion.

(4) Remove ALL the Bleach, and wash embryos 3 times with 0.1% Triton X-100. 
•Remember to wash the caps

(5) Removal ALL Triton. Then add 600uL of Heptane, followed by 600uL of Fix Solution. Fix by nutating for 20 minutes.

(!) Safety (!) Heptane and Formaldehyde are reactive to organic tissue. (!) Safety (!)
•The addition of Heptane and Fix Solution will form two liquid phase (Bottom Phase = Formaldehyde; Upper phase= Organic Heptane). 
→ Embryos should appear between the two phases.
•The Heptane is an organic molecule that pokes holes in the vitelline membrane.
→ The holes allow the Formaldehyde to access the Plasma Membrane.
•The Formaldehyde in the Fix Solution diffuses across the PM, and cross links residues (ex: Lysine).
•The PBS (Phosphate Buffered Saline) in the Fix Solution mimics physiological salt and pH levels.
→ This provides osmotic balance for the Embryo. 

NOTE: Use these 20 minutes to prepare the NGS Block Solution and 1°Antibody Solution

(6) Carefully remove the bottom Formaldehyde phase.
(!) Safety (!) Dispose the formaldehyde in the Organic Waste Container (!) Safety (!)
•Tip: Removing all the Formaldehyde at the expense of a few embryos is perfectly fine.
•Tip: Remove the phase using a p200 tip, and avoid contact with the walls.

(7) Add an equal volume of Methanol/MeOH (600uL) and shake for ~30 seconds. Afterwards, the Embryos should settle to the bottom of the tube.
•The MeOH causes “Methanol Pop”, which essentially makes small holes in the Plasma Membrane.
→ Allows us to access the inside of the cell.
•The shaking + with MeOH removes the vitelline (Devitellinization)
→ Devitellinization ensures antibody access to the permeated embryo.
•Remove embryos that remain at the interface. These are unfertilized embryos.

(8) Carefully remove ALL the liquid, and then wash the embryos 3 times with MeOH.
(!) Safety (!) Dispose the removed liquid and MeOH wash in the Organic Waste Container (!) Safety (!)
•Washing removes any residual formaldehyde.

(9) Add at least 1mL of NGS Block Solution, and nutate for 1.5 hours at Room Temperature
•NGS Block Solution contains the following compounds:
-NGS (Normal Goat Serum) contains antibodies that will bind to nonspecific sites in the embryo, allowing our primary antibodies to bind specifically to our target.
-NaN3 (Sodium Azide)→ Prevents bacterial growth
-0.1 Triton X-100 → Permeates the Plasma Membrane

(10) Remove NGS Blocking Solution. Add the 350 uL 1° Antibody Solution and nutate overnight at 4°C.

—- The Next Day —-

(11) Wash Embryos 3 times with 1X PBS or 1X PBS + 0.1% Triton. Each wash requires a ~10 min nutation. 
•This removes any excess 1° Antibody within our embryos

(12) Block again with NGS Block Solution for 30 minutes at Room Temperature

NOTE: Use these 30 minutes to prepare the 2° Antibody Solution. 

(13) Add the 400uL 2° Antibody Solution and nutate in the Dark at Room Temperature for 2 hours.

(14) Repeat the step (11)’s Wash procedure.
•This removes the residual 2° Antibodies in our embryos.

(15) Remove the supernatant, and then add 200uL of 1X PBS + 0.1% Triton Solution.
•The PBS+Triton Solution will be the medium for our transfer.→ Triton Prevents sticky embryos

(16) Using a cut p200 yellow tip, transfer the embryos on a slide in 1 drop.

(17) Remove excess liquid using the tips of a kimwipe
•Take care to not crush embryos

(18) Mount the slide using 1 drop of Aqua PolyMount solution