Try this
<html xmlns="http://www.w3.org/1999/xhtml">
<head id="Head1" runat = "server">
<title></title>
<script type = "text/javascript">
window.onload = function () {
var img1 = '<%=ResolveUrl("~/Images/asc.gif") %>';
var img2 = '<%=ResolveUrl("~/Images/desc.gif") %>';
var img = document.getElementById("<%= ImageButton1.ClientID%>");
img.src = img1;
img.onmouseover = function () {
this.src = img2;
};
img.onmouseout = function () {
this.src = img1;
};
};
</script>
</head>
<body>
<form id="form1" runat="server">
<asp:ImageButton ID="ImageButton1" runat="server" />
</form>
</body>
</html>