John Steinbeck addresses the theme of isolation and alienation in Of Mice and Men. Virtually the entire cast of characters are migrant workers which are constantly referred to as "the loneliest guys in the world"--they are isolated because of their low status in life. They have no families, no homes, and can carry all of their possessions on their backs. (The setting of the work ranch helps to emphasize this as well.)
Steinbeck does a remarkable job of showing isolation and alienation in regards to individual characters as well. Perhaps the best example of this comes in Chapter 4. Steinbeck intentionally puts Crooks, Lennie, Candy, and Curley's wife together in Crooks' room to show the reader that these are the most isolated characters. These are the ones suffering from the most alienation. Despite being outcasts of society as a whole, the other characters have gone into town together. These four characters do not go with everyone else because they are the outcasts of the outcasts. They are truly the bottom rung of the social ladder. Each of these characters has something specific which makes them fall into this category. Crooks is an outcast because of his race, Lennie because of his mental disability, Candy because of his age and physical disability (missing hand), and Curley's wife because of her gender.