We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach which makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multi-step static FAIMS mass spectrometry resulted in a six- to sixteen-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multi-step static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between two- and three-fold over single step FAIMS, and between two- and fourteen-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between two- and fourfold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue fewer total proteins were detected following heat preservation, however approximately one third were unique to the heat preserved samples.