Complete integration of all sample preparation steps in a microfluidic device greatly benefits point-of-care diagnostics. In the most simplistic approach, reagents are integrated in a microfluidic chip and dissolved upon filling with a sample fluid by capillary force. This will generally result in at least partial reagent wash-off during sample inflow. However, many applications, such as immunostaining-based cytometry, strongly rely on a homogeneous reagent distribution across the chip. The concept of initially preventing release (during inflow), followed by a triggered instantaneous and complete release on demand (after filling is completed) represents an elegant and simple solution to this problem. Here, we realize this controlled release by embedding antibodies in a gelatin layer integrated in a microfluidic chamber. The gelatin/antibody layer is deposited by inkjet printing. Maturation of this layer during the course of several weeks, due to the ongoing physical cross-linking of gelatin, slows down the antibody release, thereby reducing antibody wash-off during inflow, and consequently helping to meet the requirement for a homogeneous antibody distribution in the filled chamber. After inflow, complete antibody release is obtained by heating the gelatin layer above its sol-gel transition temperature, which causes the rapid dissolution of the entire gelatin/antibody layer at moderate temperatures. We demonstrate uniform and complete on-chip immunostaining of CD4 positive (CD4+) T-lymphocytes in whole blood samples, which is critical for accurate cell counts. The sample preparation is realized entirely on-chip, by applying temperature-switched antibody release from matured gelatin/antibody layers.