• Horseradish peroxidase + 2 H₂O₂ → O₂ + 2 H₂O
• O₂ + DAB → insoluble, brown-black precipitate

All steps performed at constant agitation on a shaker or rocking table at room temperature.

Solutions and materials:

Supermix

• TBS, 200 ml
• Gelatin, 0.5 g
• Triton X-100, 1 ml
• Heat up 5 -10 minutes to 50 – 60 ºC until gelatin dissolves and let it cool down, supermix can be stored for up to 2 weeks in the refrigerator, discard if it becomes opaque or if particles start floating which indicates the presence of fungus

TBS from stock solution or:

• dH₂O, 1 L
• Trizma HCl, 6.06 g
• Trizma Base, 1.39 g
• NaCl, 8.76 g

Prepare gelatinised glass slides

Day 1:

• Transfer sections into 12 well plate using a fine brush
• Rinse sections in TBS 4 x 10 minutes
• Incubate sections 10 minutes with blocking solution (10 % methanol, 3 % H₂O₂ in TBS)  - to prepare the solution add 1 ml 99.9 % methanol in 8 ml TBS and mix, then add 1 ml 30 % H₂O₂ and mix, H₂O₂ is used to quench endogenous peroxidase activity
• Rinse sections in TBS 4 x 10 minutes
• Incubate in the primary antibody diluted in Supermix overnight at 4 ºC

Day 2:

• Rinse sections in TBS 4 x 10 minutes
• Incubate sections in the secondary antibody diluted in Supermix 1 hour at room temperature
• Rinse sections in TBS 4 x 10 minutes
• Incubate with ABC 1 hour. ABC should be prepared more than 20 minutes  in prior to use – add 1 µl A and 1 µl B to 800 µl Supermix
• Rinse sections in TBS 4 x 10 minutes
• Incubate sections in DAB and H₂O₂ 10 minutes - dissolve 1 tablet of DAB in 10 – 15 ml PBS and mix, then add 15 µl 30 % H₂O₂ – it will produce a brown staining slowly; you may also add 0.5 ml nickel to catalyse the reaction – it will produce a deep purple or gray staining rapidly; add H₂O₂ separately when not certain about the reaction time
• Follow the reaction under microscope, rinse sections in TBS to terminate the reaction
• Sections should be stored in TBS in refrigerator until cover-slipping

Cover-slipping:

• Transfer free-floating sections onto gelatinized slides in PBS and let them dry overnight at room temperature
• Dehydrate in ethanol – 70 % ethanol 5 minutes, 95 % ethanol 5 minutes, and 100 % ethanol 10 minutes, then rinse in Xylen 20 minutes in ventilation hood
• Mount in DPX and air dry for at least 30 minutes, store at room temperature

Go is a Chinese board game invented more than three thousand years ago. First introduced to Japan and Korea and later to Europe, it has become one of the most played board games in the world. Although the basic rules were fairly simple, the challenge of its variations and complexity has made it very popular. Go has many names, its Chinese name is Wei Qi, its Japanese name is Igo and Korean name is Baduk.

The basic rules

Go is a board game for two players. The game is played on a 19 by 19 line board with 181 black stones and 180 white stones. The board has 361 intersections, each intersection makes a point, and there are nine dots on the board referred to as star points, which are used as reference points or handicap points.

Black and white stones represent each of the players. Black stones are played by the weaker part which also makes the first move. The stones are placed on the points, or intersections, on the board. After a stone has been placed on the board, it cannot be moved to another position. Each stone on the board has two to four vertically or horizontally adjacent points, with the unoccupied points named liberties. Stones that are adjacent to each other vertically or horizontally are called groups. Each group sharing common liberties and can hold more than four liberties.

When all the liberties of a stone or a group are occupied by the opponents colour, it is captured and must be removed from the board. This situation is called atari in Go term. There are also certain types of forbidden points on the board. One type is the point where a stone doesn’t have any liberties. The other is when a move shifts the stones into their previous positions. This situation is called ko. Another situation is seki, which means that black stones surrounds the white stones and in turn gets surrounded by white stones. Both colours are alive but the points inside them are excluded from the scores.

When the opponent passes his turn you have the right to decide whether the game has ended. At the end of the game, captured stones are excluded from the board. Alive stones are counted together with their surrounded points minus the number of captured stones. Weak players may also use handicap, i.e. place their stones on the star points at the beginning. If handicap is applied, certain points must be reduced from the score. Since black makes the first move it is considered as a handicap and 6.5 points are added to white. This situation is called komi.

Basic strategies

The goal of the game is to acquire territories. You may not play the stones only by tackle a local conflict that appears on the board; you must also consider how to expand you territories, i.e. the points surrounded inside groups. Having the initiative gives you more control over the board. Sometime it is worth to sacrifice a stone in order to regain the initiative. You must also avoid dead stones or dean groups. This can be accomplished by creating eyes, which are unoccupied points formed by a group connected in a chain. The eyes can never be captured and makes living groups. One of the most effective ways of taking initiative is to cut the opponents groups into smaller ones, because smaller groups are easier to capture. Thus, getting your groups connected will give you more advantage.

Tournament

A game starts with the white player grabs some stones and asks the opponent to guess whether the number of stones in the hand is odd or even. If the opponent answers correctly, he will choose the colour. Each player is given 1.5 hours time total and you must end the game before the time is used up, otherwise you will lose. A player starts his move by pressing the clock and ends the move in the same way. Go players are ranked from beginners called kyu to professional called dan. Kyu is ranked from 30 kyu to 1 kyu and dan is ranked from 1 dan to 9 dan. The rules of scoring are different in Chinese go and Japanese go. In Chinese rules all the captured stoned must be counted and added to the opponents score, while in Japanese rules only the territories are counted.

Comments

Go is an intellectual challenging game because it offers innumerable combinations. Playing Go may develop you mathematical skills and improve you memory. There are many books about Go that describe more advanced strategies and tricks. You may be able to memorize a good deal of these rules but the essence is how to apply them on the board. To become a professional, you need practice and learn to be flexible. However, if you have decided to become a Go player but cannot find an opponent immediately, you can download computer software from www.britgo.org.

by Ting on June 1, 2004