I do not understand very well what you mean by unchecking, I'm going to give you an example with what I understand of your question.
When you click on a button you perform an action, in this case that button changes color, I also want the rest of the buttons to return to their original color, that is, I just want to have an active button.
For this I'm going to create a generic method.
This method will go through all my ToolStrip in search of buttons whose name is the same as the one I have clicked, that is, the active one.
private void Desmarca(string buttonName)
//Recorremos el toolstrip en busca de botones
foreach (ToolStripButton boton in this.toolStrip1.Items)
// Si el boton no es el mismo que ha sido clickado
if (boton.Name != buttonName)
//Tu acción(desmarcar), en mi caso cambiar el color
boton.BackColor = Color.Green;
Now what remains to be done is to add to each button the code to call this fragment.
private void toolStripButton2_Click(object sender, EventArgs e)
//Casteo del sender a objeto de botón
ToolStripButton B = (ToolStripButton)sender;
//Tu acción, en mi caso cambiar el color
B.BackColor = Color.Yellow;
//Llamada al fragmento que controla el resto de botones
I hope it can help you. Do not hesitate to clarify your situation in case I can be more specific in your problem.